Phosphodiesterase 4d7 as prostate cancer marker

ABSTRACT

The present invention relates to phosphodiesterase 4D7 (PDE4D7) for use as a marker for prostate cancer, and the use of PDE4D7 as a marker for diagnosing, detecting, monitoring or prognosticating prostate cancer or the progression of prostate cancer. The present invention also relates to a composition for diagnosing, detecting, monitoring or prognosticating prostate cancer or the progression of prostate cancer, a corresponding method and immunoassay, a method for diagnosing, monitoring or prognosticating hormone-resistant prostate cancer vs. hormone-sensitive prostate cancer, a corresponding immunoassay, a method of data acquisition, an immunoassay for diagnosing, detecting, monitoring or prognosticating prostate cancer or the progression of prostate cancer, a method of identifying an individual for eligibility for prostate cancer therapy, an immunoassay for stratifying an individual or cohort of individuals with a prostate cancer disease, an immunoassay for stratifying an individual with prostate cancer, as well as a pharmaceutical composition comprising a compound directly stimulating or modulating the activity of PDE4D7, a compound indirectly stimulating or modulating the activity of PDE4D7, the PDE4D7 protein or a biologically active equivalent thereof, a nucleic acid encoding and expressing PDE4D7, a miRNA inhibitor specific for PDE4D7 miRNAs, a demethylation agent and/or a phosphodiesterase displacement factor.

FIELD OF THE INVENTION

The present invention relates to phosphodiesterase 4D7 (PDE4D7) for useas a marker for prostate cancer, and the use of PDE4D7 as a marker fordiagnosing, detecting, monitoring or prognosticating prostate cancer orthe progression of prostate cancer. The present invention also relatesto a composition for diagnosing, detecting, monitoring orprognosticating prostate cancer or the progression of prostate cancer, acorresponding method and immunoassay, a method for diagnosing,monitoring or prognosticating hormone-resistant prostate cancer vs.hormone-sensitive prostate cancer, a corresponding immunoassay, a methodof data acquisition, an immunoassay for diagnosing, detecting,monitoring or prognosticating prostate cancer or the progression ofprostate cancer, a method of identifying an individual for eligibilityfor prostate cancer therapy, an immunoassay for stratifying anindividual or cohort of individuals with a prostate cancer disease, animmunoassay for stratifying an individual with prostate cancer, as wellas a pharmaceutical composition comprising a compound directlystimulating or modulating the activity of PDE4D7, a compound indirectlystimulating or modulating the activity of PDE4D7, the PDE4D7 protein ora biologically active equivalent thereof, a nucleic acid encoding andexpressing PDE4D7, a miRNA inhibitor specific for PDE4D7 miRNAs, ademethylation agent and/or a phosphodiesterase displacement factor.

BACKGROUND OF THE INVENTION

Cancer is a class of diseases in which a group of cells displayuncontrolled growth, invasion and sometimes metastasis. These threemalignant properties of cancers differentiate them from benign tumors,which are self-limited, do not invade or metastasize. Among men, thethree most commonly diagnosed cancers are prostate, lung and colorectalcancer in developed countries. Particularly prostate cancer is the mostcommon malignancy in European males. In 2002 in Europe, an estimated225,000 men were newly diagnosed with prostate cancer and about 83,000died from this disease.

Certain phosphodiesterases have been associated with cancer development.For instance, phosphodiesterase PDE7 has been shown to be linked tochronic lymphocytic leukemia (Zhang L et al., PNAS, 2008, 105(49):19532-7). Yet, for many cancer types or cancer progression forms thereis no adequate marker molecule available.

Prostate cancer, for example, is traditionally diagnosed via the serumlevel of prostate-specific antigen (PSA). However, PSA is not prostatecancer-specific and can be raised in other circumstances, leading to alarge number of false-positives (cancer is not found in around 70% ofmen with raised PSA levels who undergo biopsy). Furthermore, there willbe an unpredictable number of false-negatives who later develop prostatecancer in the presence of a “normal” PSA test.

Therefore, there is a need for the provision of a new and effective,alternative diagnosis perspective for the detection, monitoring andprognostication of prostate cancer.

SUMMARY OF THE INVENTION

The present invention addresses this need and provides means and methodswhich allow the diagnosis and detection of prostate cancer.

The above objective is accomplished by phosphodiesterase 4D7 (PDE4D7)for use as a prostate cancer marker.

Phosphodiesterase 4D7 is shown by the present inventors to bedown-regulated in prostate cancer cell lines and patient derivedprostate tissue. PDE4D7 is, thus, considered as a biomarker for prostatecancer prediction and a decision tool for the stratification of certaincancer surveillance regimes, as well as the prognosis and monitoring ofprostate cancer progression. In particular, it was demonstrated by thepresent inventors that PDE4D7 is down-regulated in hormone-resistanthuman-derived prostate cell lines as well as corresponding human tissuesamples. Diagnostic methods and uses based on PDE4D7 as a prostatecancer marker can, thus, advantageously be employed for (i) detectingand diagnosing life-threatening prostate cancer forms, (ii)prognosticating life-threatening prostate cancer forms, (iii) monitoringof cancer progression towards life-threatening prostate cancer forms,and (iv) distinguishing between indolent and life-threatening cancerforms.

In another aspect the present invention relates to a composition fordiagnosing, detecting, monitoring or prognosticating prostate cancer orthe progression of prostate cancer, comprising a nucleic acid affinityligand and/or a peptide affinity ligand for the PDE4D7 expressionproduct or protein.

In a preferred embodiment of the present invention said compositioncomprises a nucleic acid affinity ligand or peptide affinity ligandwhich is modified to function as a contrast agent.

In a further preferred embodiment of the present invention saidcomposition comprises a set of oligonucleotides specific for the PDE4D7expression product, a probe specific for the PDE4D7 expression product,an aptamer specific for the PDE4D7 expression product or protein, anantibody specific for the PDE4D7 protein and/or an antibody variantspecific for the PDE4D7 protein.

In a further aspect the present invention relates to the use of PDE4D7as a marker for diagnosing, detecting, monitoring or prognosticatingprostate cancer or the progression of prostate cancer.

In another aspect the present invention relates to a method fordetecting, diagnosing, monitoring or prognosticating prostate cancer orthe progression of prostate cancer comprising the step of determiningthe level of PDE4D7.

In another aspect the present invention relates to a method fordiagnosing, monitoring or prognosticating hormone-resistant prostatecancer or the progression towards hormone-resistant prostate cancer,wherein said method discriminates between a hormone-sensitive and ahormone-resistant prostate cancer, comprising the steps of

(a) determining the level of PDE4D7 in a sample;

(b) determining the level of expression of a reference gene in a sample;

(c) normalizing the measured expression level of PDE4D7 to theexpression of the reference gene; and

comparing the normalized expression level with a predetermined cutoffvalue chosen to exclude hormone-sensitive prostate cancer, wherein anormalized expression level below the cutoff value is indicative of ahormone-resistant prostate cancer, wherein said cutoff value is betweenabout 1 and 7, preferably about 5.

In another aspect the present invention relates to a method of dataacquisition comprising at least the steps of:

(a) testing in an individual for expression of PDE4D7; and

(b) comparing the expression as determined in step (a) to a controllevel.

In a further preferred embodiment of the present invention thediagnosing, detecting, monitoring prognosticating or data acquisition isto be carried out on a sample obtained from an individual.

In another aspect the present invention relates to an immunoassay fordetecting, diagnosing, monitoring or prognosticating prostate cancer orthe progression of prostate cancer comprising at least the steps of:

(a) testing in a sample for the expression of PDE4D7,

(b) testing in a control sample for the expression of PDE4D7,

(c) determining the difference in expression of PDE4D7 of steps (a) and(b); and

(d) deciding on the presence or stage of prostate cancer or theprogression of prostate cancer based on the results obtained in step(c),

wherein said testing steps are based on the use of an antibodyspecifically binding to PDE4D7.

In another aspect the present invention relates to an immunoassay fordiscriminating between a hormone-sensitive and a hormone-resistantprostate cancer, comprising the steps of:

(a) determining the level of PDE4D7 in a sample;

(b) determining the level of expression of a reference gene in a sample;

(c) normalizing the measured expression level of PDE4D7 to theexpression of the reference gene; and

(d) comparing the normalized expression level with a predeterminedcutoff value to exclude hormone-sensitive prostate cancer, wherein anormalized expression level below the cutoff value is indicative of ahormone-resistant prostate cancer, wherein said cutoff value is betweenabout 1 and 7, preferably about 5.

In another aspect the present invention relates to a method ofidentifying an individual for eligibility for prostate cancer therapycomprising:

(a) testing in a sample obtained from an individual for the expressionof PDE4D7;

(b) testing in said sample for the expression of a reference gene and/ortesting in a control sample for the expression of PDE4D7;

(c) classifying the levels of expression of step (a) relative to levelsin control samples of PDE4D7 of step (b); and

(d) identifying the individual as eligible to receive a prostate cancertherapy where the individual's sample is classified as having a reducedlevel of PDE4D7 expression.

In yet another aspect the present invention relates to an immunoassayfor stratifying an individual or cohort of individuals with a prostatecancer disease comprising:

(a) testing in a sample obtained from an individual for the expressionof PDE4D7;

(b) testing in said sample for the expression of a reference gene and/ortesting in a control sample for the expression of PDE4D7;

(c) determining the difference in expression of PDE4D7 of step (a) andthe expression of PDE4D7 and/or the reference gene in step (b); and

(d) stratifying an individual or cohort of individuals to prostatecancer therapy based on the results obtained in step (c), where theindividual's sample has a reduced level of PDE4D7 expression.

In a further preferred embodiment of the present invention said testingor determining of the expression is accomplished, or additionallyaccomplished, by the measurement of nucleic acid or protein levels or bythe determination of the biological activity of PDE4D7, or of thereference gene.

In a further preferred embodiment of the present invention said methodor immunoassay comprises the additional step of comparing the measurednucleic acid or protein levels or the measured biological activity to acontrol level.

In a further preferred embodiment of the present invention saidreference gene is a housekeeping gene, particularly preferred GAPDH orPBGD, or a different phosphodiesterase, particularly preferred PDE4D5.

In a further preferred embodiment of the present invention said methodor immunoassay comprises the additional step of determining the level ofprostate specific antigen (PSA).

In a further preferred embodiment of the present invention the sample asmentioned above is a tissue sample, a urine sample, a urine sedimentsample, a blood sample, a saliva sample, a semen sample, a samplecomprising circulating tumor cells or a sample containing prostatesecreted exosomes.

In yet another aspect the present invention relates to a pharmaceuticalcomposition comprising at least one element selected from the group of:

(a) a compound directly stimulating or modulating the activity ofPDE4D7, preferably an allosteric agonist of PDE4D7 enzymatic activity;

(b) a compound indirectly stimulating or modulating the activity ofPDE4D7;

(c) the PDE4D7 protein or a biologically active equivalent thereof;

(d) a nucleic acid encoding and expressing PDE4D7;

(e) a miRNA inhibitor specific for PDE4D7 miRNAs;

(f) a demethylation agent; and

(g) a phosphodiesterase displacement factor.

As phosphodiestrase 4D7 is down-regulated in disease-associated celllines, PDE4D7 itself and agents modifying PDE4D7, modifying PDE4D7expression or modifying PDE4D7 interactions can advantageously be usedas medicaments. Thus, by counteracting the observed down-regulationprocess, PDE4D7 and/or PDE4D7 modification agents may be used as amedicament, e.g. as a medicament counteracting all or some of theeffects associated with a low PDE4D7 expression or its down-regulation.

In a further aspect the present invention relates to a pharmaceuticalcomposition for the treatment or prevention of prostate cancercomprising at least one element selected from the group of:

(a) a compound directly stimulating or modulating the activity ofPDE4D7, preferably an allosteric agonist of PDE4D7 enzymatic activity;

(b) a compound indirectly stimulating or modulating the activity ofPDE4D7;

(c) the PDE4D7 protein or a biologically active equivalent thereof;

(d) a nucleic acid encoding and expressing PDE4D7;

(e) a miRNA inhibitor specific for PDE4D7 miRNAs;

(f) a demethylation agent; and

(g) a phosphodiesterase displacement factor.

As phosphodiesterase 4D7 is down-regulated in cancer cell lines, PDE4D7itself and agents modifying PDE4D7 or modifying PDE4D7 expression ormodifying PDE4D7 interactions can advantageously be used as medicamentsfor the treatment of cancer, in particular for the treatment of prostatecancer. Thus, by counteracting the observed down-regulation process,PDE4D7 and/or PDE4D7 modification agents may be used as a medicamentcounteracting the low PDE4D7 expression and/or the PDE4D7down-regulation in cancerous cells, in particular in prostate cancercells.

In a preferred embodiment of the present invention the prostate cancertherapy as mentioned herein above comprises the administration of thepharmaceutical composition as defined herein above, or theadministration of the pharmaceutical composition as defined herein abovein combination with an additional cancer therapy, preferably radiationtherapy or chemotherapy.

In another aspect the present invention relates to a method of treatmentor prevention of cancer, in particular prostate cancer, comprising theadministration of

(a) a compound directly stimulating or modulating the activity ofPDE4D7, preferably an allosteric agonist of PDE4D7 enzymatic activity;

(b) a compound indirectly stimulating or modulating the activity ofPDE4D7;

(c) the PDE4D7 protein or a biologically active equivalent thereof;

(d) a nucleic acid encoding and expressing PDE4D7;

(e) a miRNA inhibitor specific for PDE4D7 miRNAs;

(f) a demethylation agent; and/or

(g) a phosphodiesterase displacement factor to an individual.

In a preferred embodiment of the present invention saidphosphodiesterase displacement factor as mentioned above is a peptide, apeptidomimetic, a small molecule, an antibody or an aptamer.

In yet another preferred embodiment of the present invention saidprostate cancer is hormone-resistant prostate cancer.

These and other characteristics, features and objectives of the presentinvention will become apparent from the following detailed description,taken in conjunction with the accompanying figures and examples, whichdemonstrate by way of illustration the principles of the invention.

The description is given for the sake of example only, without limitingthe scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 gives an overview over the samples tested on expression levels.AD means “androgen dependent”, AS stands for “androgen sensitive” and AImeans “androgen independent”. Samples “PC346P xenograft” through“346Flu2” are cell lines, samples “PC295” through “PC374” arexenografts.

FIG. 2 depicts the relative PDE4D7 mRNA expression in several prostatecancer cell lines and xenografts normalized to GAPDH compared to LNCaP.The figure provides an overview over all investigated samples, includingcell lines and xenograft material.

FIG. 3 depicts the relative PDE4D7 mRNA expression in prostate cancercell lines normalized to GAPDH compared to LNCaP.

FIG. 4 depicts the relative PDE4D7 mRNA expression in prostate cancerxenografts normalized to GAPDH compared to LNCaP.

FIG. 5 shows the PDE4D7 content expressed as a percentage of total PDE4DmRNA in prostate cancer cell lines and xenografts. The figure providesan overview over all investigated samples, including cell lines andxenograft material.

FIG. 6 shows the PDE4D7 content expressed as a percentage of total PDE4DmRNA in prostate cancer cell lines.

FIG. 7 shows the PDE4D7 content expressed as percentage of total PDE4DmRNA in prostate cancer xenografts.

FIG. 8 shows the relative gene expression of human PDE4D7 in humanpatient tissue samples. Information is derived from 16 different samplesin total, as depicted in Table 1 (including the lymph node resectedtissues). Sample group “1=no” is defined as hormone-responsive primaryprostate tumors, Sample group “2=yes” is defined as hormone-resistantprostate tumors. Indicated are the individual relative expression valuesfor human PDE4D7 on human prostate tissues. The results were normalizedto the expression of GAPDH and PBGD. The median of the data relativedata measurements is indicated for each patient group.

FIG. 9 shows the relative gene expression of human PDE4D7 in humanpatient tissue samples. Information is derived from 16 different samplesin total, as depicted in Table 1 (including the lymph node resectedtissues). Sample group “1” is defined as hormone-responsive primaryprostate tumors, Sample group “2” is defined as hormone-resistantprostate tumors. The results were normalized to the expression of GAPDHand PBGD. The figure shows a box plot of the individual data relativeexpression measurements for human PDE4D7, whereby the box includes 75%of all measurements. The median relative expression value is indicatedas the border between the two colored boxes.

FIG. 10 shows the relative gene expression of human PDE4D7 in humanpatient tissue samples. Information is derived from 12 different samplesin total, as depicted in Table 1 (excluding the lymph node resectedtissues). Sample group “1=no” is defined as hormone-responsive primaryprostate tumors, Sample group “2=yes” is defined as hormone-resistantprostate tumors. The results were normalized to the expression of GAPDHand PBGD. Indicated are the individual relative expression values forhuman PDE4D7 on human prostate tissues. The median of the data relativedata measurements is given for each patient group.

FIG. 11 shows the relative gene expression of human PDE4D7 in humanpatient tissue samples. Information is derived from 12 different samplesin total depicted in Table 1 (excluding the lymph node resectedtissues). Sample group “1” is defined as hormone-responsive primaryprostate tumors, Sample group “2” is defined as hormone-resistantprostate tumors. The results were normalized to the expression of GAPDHand PBGD. The figure shows a box plot of the individual data relativeexpression measurements for human PDE4D7, whereby the box includes 75%of all measurements. The median relative expression value is indicatedas the border between the two colored boxes.

FIG. 12 shows the effect of human PDE4D7 in vivo expression on theproliferation of PC3 prostate cancer cells.

DETAILED DESCRIPTION OF EMBODIMENTS

The inventors have found that PDE4D7 is strongly down-regulated incertain prostate cancer-associated cell types and human patient tissuesand can, hence, be used as biomarker for prostate cancer. PDE4D7 as wellas agents modifying PDE4D7 or modifying PDE4D7 expression can further beused as medicaments, in particular for the treatment of prostate cancer.

Although the present invention will be described with respect toparticular embodiments, this description is not to be construed in alimiting sense.

Before describing in detail exemplary embodiments of the presentinvention, definitions important for understanding the present inventionare given.

As used in this specification and in the appended claims, the singularforms of “a” and “an” also include the respective plurals unless thecontext clearly dictates otherwise.

In the context of the present invention, the terms “about” and“approximately” denote an interval of accuracy that a person skilled inthe art will understand to still ensure the technical effect of thefeature in question. The term typically indicates a deviation from theindicated numerical value of ±20%, preferably ±15%, more preferably±10%, and even more preferably ±5%.

It is to be understood that the term “comprising” is not limiting. Forthe purposes of the present invention the term “consisting of” isconsidered to be a preferred embodiment of the term “comprising of”. Ifhereinafter a group is defined to comprise at least a certain number ofembodiments, this is meant to also encompass a group which preferablyconsists of these embodiments only.

Furthermore, the terms “first”, “second”, “third” or “(a)”, “(b)”,“(c)”, “(d)” etc. and the like in the description and in the claims, areused for distinguishing between similar elements and not necessarily fordescribing a sequential or chronological order. It is to be understoodthat the terms so used are interchangeable under appropriatecircumstances and that the embodiments of the invention described hereinare capable of operation in other sequences than described orillustrated herein.

In case the terms “first”, “second”, “third” or “(a)”, “(b)”, “(c)”,“(d)” etc. relate to steps of a method or use there is no time or timeinterval coherence between the steps, i.e. the steps may be carried outsimultaneously or there may be time intervals of seconds, minutes,hours, days, weeks, months or even years between such steps, unlessotherwise indicated in the application as set forth herein above orbelow.

It is to be understood that this invention is not limited to theparticular methodology, protocols, proteins, bacteria, vectors, reagentsetc. described herein as these may vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention that will be limited only by the appended claims.Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art.

As has been set out above, the present invention concerns in one aspectphosphodiesterase 4D7 (PDE4D7) for use as a prostate cancer marker. Theterm “phosphodiesterase 4D7” or “PDE4D7” relates to the splice variant 7of the human phosphodiesterase PDE4D, i.e. the human phosphodiesterasePDE4D7 gene, preferably to the sequence as defined in Genbank AccessionNo: AF536976 (version AF536976.1, GI:22901883 as of 3 Mar. 2009), morepreferably to the nucleotide sequence as set forth in SEQ ID NO: 1,which corresponds to the sequence of the above indicated GenbankAccession number of the PDE4D7 transcript, and also relates to thecorresponding amino acid sequence as set forth in SEQ ID NO: 2, whichcorresponds to the sequence of the above indicated Genbank Accessionnumber of the PDE4D7 polypeptide encoded by the PDE4D7 transcript. Theterm also comprises nucleotide sequences showing a high degree ofhomology to PDE4D7, e.g. nucleic acid sequences being at least 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to thesequence as set forth in SEQ ID NO: 1, or amino acid sequences being atleast 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to the sequence as set forth in SEQ ID NO: 2, or nucleic acidsequences encoding amino acid sequences being at least 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to thesequence as set forth in SEQ ID NO: 2, or amino acid sequences beingencoded by nucleic acid sequences being at least 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequenceas set forth in SEQ ID NO: 1.

The term “human phosphodiesterase PDE4D7 gene”, “PDE4D7 gene” or “PDE4D7marker gene” as used herein relates to the gene encodingphosphodiesterase 4D. Preferably, the term relates to a gene expressingphosphodiesterase 4D as splice variant 7, e.g. the specific exoncombination as defined in Genbank Accession No: AF536976 (versionAF536976.1, GI:22901883 as of 3 Mar. 2009) or as set forth in SEQ IDNO: 1. The term also relates to DNA molecules derived from mRNAtranscripts encoding phosphodiesterase 4D spliced as variant 7,preferably cDNA molecules.

The term “marker” or “PDE4D7 marker”, as used herein, relates to a gene,genetic unit or sequence (a nucleotide sequence or amino acid or proteinsequence) as defined herein above, whose expression level is modified,preferably decreased, in a cancerous cell or in cancerous tissue or inany type of sample comprising cancerous cells or cancerous tissues orportions or fragments thereof, in comparison to a control level orstate. The term also refers to any expression product of said geneticunit or sequence, in particular to a PDE4D7 mRNA transcript, apolypeptide encoded by the PDE4D7 transcript or variants or fragmentsthereof, as well as homologous derivatives thereof as described hereinabove. The term “expression level” as used herein refers to the amountof PDE4D7 transcript and/or PDE4D7 protein derivable from a definednumber of cells or a defined tissue portion, preferably to the amount ofPDE4D7 transcript and/or PDE4D7 protein obtainable in a standard nucleicacid (e.g. RNA) or protein extraction procedure. Suitable extractionmethods are known to the person skilled in the art.

The term “control level” (or “control state”), as used herein, relatesto an expression level which may be determined at the same time and/orunder similar or comparable conditions as the test sample by using (a)sample(s) previously collected and stored from a subject/subjects whosedisease state, e.g. non-cancerous, is/are known. The term “diseasestate” or “cancerous disease state” relates to any state or type ofcellular or molecular condition between a non-cancerous cell state and(including) a terminal cancerous cell state. Preferably, the termincludes different cancerous proliferation/developmental stages orlevels of tumor development in the organism between (and excluding) anon-cancerous cell state and (including) a terminal cancerous cellstate. Such developmental stages may include all stages of the TNM(Tumor, Node, Metastasis) classification system of malignant tumors asdefined by the UICC, e.g. stages 0 and I to IV. The term also includesstages before TNM stage 0, e.g. developmental stages in which cancerbiomarkers known to the person skilled in the art show a modifiedexpression or expression pattern.

The expression level as mentioned above may preferably be the expressionlevel of PDE4D7 as defined herein above. Alternatively or additionally,the expression level may also be the expression level of any othersuitable gene or genetic element expressed in a cell, preferably in thecontext of PDE4D7, e.g. the expression level of anotherphosphodiesterase, the expression level of a housekeeping gene, e.g.GAPDH or PBGD.

The term “cancerous” relates in the context of the present invention toa cancerous disease state as defined herein above. A preferred controllevel in the context of cancerous controls is the expression of PDE4D7in malignant, hormone-sensitive tumors.

The term “non-cancerous” relates in the context of the present inventionto a condition in which neither benign nor malign proliferation can bedetected. Suitable means for said detection are known in the art. Apreferred control level in the context of non-cancerous controls is theexpression of PDE4D7 in normal, i.e. healthy or non-cancerous tissue orthe expression of PDE4D7 in benign prostate tumor tissue. The term“benign prostate tumor” as used herein refers to a prostate tumor whichlacks all three of the malignant properties of a cancer, i.e. does notgrow in an unlimited, aggressive manner, does not invade surroundingtissues, and does not metastasize. Typically, a benign prostate tumorimplies a mild and non-progressive prostate neoplastic or swellingdisease lacking the invasive properties of a cancer. Furthermore, benignprostate tumors are typically encapsulated, and thus inhibited in theirability to behave in a malignant manner. A benign tumor or a healthycondition may be determined by any suitable, independent molecular,histological or physiological method known to the person skilled in theart.

Alternatively, the control level may be determined by a statisticalmethod based on the results obtained by analyzing previously determinedexpression level(s) of the PDE4D7 marker gene of the present inventionin samples from subjects whose disease state is known. Furthermore, thecontrol level can be derived from a database of expression patterns frompreviously tested subjects or cells. Moreover, the expression level ofthe marker genes of the present invention in a biological sample to betested may be compared to multiple control levels, whose control levelsare determined from multiple reference samples. It is preferred to use acontrol level determined from a reference sample derived from a tissuetype similar to that of the patient-derived biological sample. It isparticularly preferred to use sample(s) derived from a subject/subjectswhose disease state is non-cancerous as defined herein above, i.e. whichpresent a health condition in which neither benign nor malignproliferation can be detected. In another embodiment of the presentinvention, the control level can be determined from a reference samplederived from a subject who has been diagnosed to suffer from prostatecancer, e.g. from hormone-independent or hormone-resistant prostatecancer.

Alternatively, reference samples may comprise material derived from celllines, e.g. immortalized cancer cell lines, or be derived from tissuexenografts. Preferably, material derived from prostate cancer cell linesor material derived from tissue xenografts with human prostate tissue,in particular with benign and tumor-derived human prostate tissue, maybe comprised in a reference sample according to the present invention.Examples of preferred cancer cell lines comprise cells lines PC346P,PC346B, LNCaP, VCaP, DuCaP, PC346C, PC3, DU145, PC346CDD, PC346Flu1,PC346Flu2. Examples of preferred xenografts comprise PC295, PC310,PC-EW, PC82, PC133, PC135, PC324 and PC374. Preferably an entire panelof cell lines and xenografts may be used, e.g. the human PC346 panel.Further preferred are cell lines and xenografts as described in Marqueset al., 2006, Eur. Urol., 49(2):245-57.

In a further, preferred alternative, reference samples may be derivedfrom patient tissues, or tissue panels or tissue collections obtained inclinical environments. The samples may, for example, be obtained frommale patients undergoing surgery. The samples may be derived from anysuitable tissue type, e.g. from prostate tissue or lymph nodes.Preferred examples of patient tissue collections are from surgicalprostate procedures (e.g., prostatectomy).

Moreover, it is preferred to use the standard value of the expressionlevels of the PDE4D7 marker of the present invention in a populationwith a known disease state. The standard value may be obtained by anymethod known in the art. For example, a range of mean±2 SD (standarddeviation) or mean±3 SD may be used as standard value.

Furthermore, the control level may also be determined at the same timeand/or under similar or comparable conditions as the test sample byusing (a) sample(s) previously collected and stored from asubject/subjects whose disease state is/are known to be cancerous, i.e.who have independently been diagnosed to suffer from a certain cancertype, e.g. from prostate cancer, in particular hormone-dependent,hormone-sensitive or hormone-resistant prostate cancer.

In the context of the present invention, a control level determined froma biological sample that is known not to be cancerous is called “normalcontrol level”. If the control level is determined from a cancerousbiological sample, e.g. a sample from a subject for which prostatecancer, in particular hormone-dependent, hormone-sensitive orhormone-resistant cancer was diagnosed independently, it may bedesignated as “cancerous control level”.

The term “prostate cancer” relates to a cancer of the prostate gland inthe male reproductive system, which occurs when cells of the prostatemutate and begin to multiply out of control. Typically, prostate canceris linked to an elevated level of prostate-specific antigen (PSA). Inone embodiment of the present invention the term “prostate cancer”relates to a cancer showing PSA levels above 4.0. In another embodimentthe term relates to cancer showing PSA levels above 2.0. The term “PSAlevel” refers to the concentration of prostate specific antigen (PSA) inthe blood in ng/ml.

The term “hormone-dependent prostate cancer” means that the growthand/or proliferation of prostate cancer or prostate cancer cell lines isdependent on male sex hormone stimulation.

The term “hormone-sensitive prostate cancer” means that the growth andproliferation of prostate cancer or prostate cancer cell lines issensitive on male sex hormone stimulation. The term “sensitive” relatesto situations in which the prostate cancer or prostate cancer cell lineshows a biochemical or cellular reaction pattern in the presence of malesex hormones, but does need a male sex hormone for growth and/orproliferation.

The term “hormone-resistant prostate cancer” means that the growth andproliferation of prostate cancer or prostate cancer cell lines isresistant to male sex hormone stimulation. The term also relates to alate prostate cancer developmental stage which is no longer amenable toan administration of anti-hormones, preferably anti-androgens as definedherein above. The term “male sex hormone” as used herein refers to anandrogen, preferably to testosterone, androstenedione,dihydrotestosterone, dehydroepiandrosterone, androstenediol orandrosterone.

In a further aspect the present invention relates to the use of PDE4D7as a marker for diagnosing, detecting, monitoring or prognosticatingprostate cancer or the progression of prostate cancer.

The term “diagnosing prostate cancer” as used herein means that asubject or individual may be considered to be suffering from prostatecancer, when the expression level of the PDE4D7 marker of the presentinvention is modified, preferably reduced or down-regulated, compared toa control level as defined herein above, preferably if compared to thenormal control level as defined herein above. The term “diagnosing” alsorefers to the conclusion reached through that comparison process.

The term “modified” or “modified expression level” in the context of thepresent invention thus denotes a change in the expression level.Expression levels are deemed to be “changed” when the PDE4D7 geneexpression, e.g. in a sample to be analyzed, differs by, for example,5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more than 50%from a control level, or at least 0.1 fold, at least 0.2 fold, at least1 fold, at least 2 fold, at least 5 fold, or at least 10 fold or more incomparison to a control level. The control level may either be a normalcontrol level or a cancerous control level as defined herein above. If acomparison with a cancerous control level is to be carried out, anadditional comparison with a normal control level is preferred. Such anadditional comparison allows for the determination of a tendency of themodification, i.e. an increase or a decrease of the expression level isobserved.

The term “modified” relates preferably to a decrease or down-regulationof the expression level of the PDE4D7 marker or a complete inhibition ofthe PDE4D7 marker expression if a test sample is compared to a controllevel. The control level may either be a normal control level or acancerous control level as defined herein above. In a preferredembodiment of the present invention the control level is a cancerouscontrol level derived from, or associated with hormone-dependentprostate tumors or tissues, more preferably derived from or associatedwith hormone-sensitive prostate tumors or tissues. The term “reducedexpression level” or “down-regulated expression level” or “decrease ofexpression level” (which may be used synonymously) in the context of thepresent invention thus denotes a reduction of the expression level ofPDE4D7 between a situation to be analyzed, e.g. a situation derivablefrom a patient's sample, and a reference point, which could either be anormal control level or cancerous control level derivable from anysuitable cancer stage known to the person skilled in the art, preferablya hormone-dependent, more preferably a hormone-sensitive prostate tumorstage. Expression levels are deemed to be “reduced” or “down-regulated”when the PDE4D7 gene expression decreases by, for example, 5%, 6%, 7%,8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more than 50% from acontrol level, or at least 0.1 fold, at least 0.2 fold, at least 1 fold,at least 2 fold, at least 5 fold, or at least 10 fold or more incomparison to a control level, preferably in comparison to ahormone-dependent, or to a hormone-sensitive prostate tumor control.

In a further embodiment, an additional similarity in the overall geneexpression pattern between a sample obtained from a subject and areference as defined herein above, which is cancerous, indicates thatthe subject is suffering from a cancer. In another embodiment of thepresent invention, the diagnosis may be combined with the elucidation ofadditional cancer biomarker expression levels. For example, theexpression of biomarkers like PSA may be tested.

A cancer, in particular a prostate cancer, may be considered as beingdiagnosed when the expression level of the PDE4D7 marker of the presentinvention is modified, preferably reduced or down-regulated, compared toa control level as defined herein above, e.g. the normal control levelas defined herein above.

In a particularly preferred embodiment a prostate cancer may consideredas being diagnosed if the PDE4D7 expression level, as defined hereinabove, is decreased by a value of between 20% to 80%, preferably by avalue of 30%, 40%, 50%, 60% or 70% in a test sample in comparison to acontrol level, preferably in comparison to a control expression levelderived from a hormone-dependent tumor control or a hormone-sensitiveprostate tumor control. In a further preferred embodiment ahormone-resistant prostate cancer may be considered as being diagnosedif the PDE4D7 expression level, as defined herein above, is decreased bya value of between 20% to 90%, preferably by a value of 30%, 40%, 50%,60%, 70% or 80% in a test sample in comparison to a control level. Thecontrol level may either be a normal control level or a cancerouscontrol level, preferably derivable from a hormone-dependent orhormone-sensitive prostate cancer.

The term “detecting prostate cancer” as used herein means that thepresence of a cancerous disease or disorder in an organism may bedetermined or that a cancerous disease or disorder may be identified inan organism. The determination or identification of a cancerous diseaseor disorder may be accomplished by a comparison of the expression levelof the PDE4D7 marker of the present invention and the normal controllevel as defined herein above. A cancer, in particular a prostatecancer, may be detected when the expression level of the PDE4D7 markeris similar to a cancerous control level as defined herein above. In apreferred embodiment of the present invention a prostate cancer may bedetected if the expression level of the PDE4D7 marker is similar to acancerous control level of an established prostate cancer cell or cellline, e.g. a prostate cancer cell line as mentioned herein above.

The term “monitoring prostate cancer” as used herein relates to theaccompaniment of a diagnosed or detected cancerous disease or disorder,e.g. during a treatment procedure or during a certain period of time,typically during 2 months, 3 months, 4 months, 6 months, 1 year, 2years, 3 years, 5 years, 10 years, or any other period of time. The term“accompaniment” means that states of disease as defined herein aboveand, in particular, changes of these sates of disease may be detected bycomparing the expression level of the PDE4D7 marker of the presentinvention in a sample to a normal or a cancerous control level asdefined herein above, preferably a control expression level derived froma hormone-dependent tumor control or a hormone-sensitive prostate tumorcontrol in any type of periodical time segment, e.g. every week, every 2weeks, every month, every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 month,every 1.5 year, every 2, 3, 4, 5, 6, 7, 8, 9 or 10 years, during anyperiod of time, e.g. during 2 weeks, 3 weeks, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12 months, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 years,respectively. The cancerous control level may be derived from samplescorresponding to different stages of cancer development, e.g. stages 0and I to IV of the TNM classification system. In a preferred embodimentof the present invention the term relates to the accompaniment of adiagnosed prostate cancer, more preferably of a hormone-dependent and ahormone-sensitive prostate cancer. In a further embodiment themonitoring may also be used for the accompaniment of hormone-resistantprostate cancer, e.g. during a treatment procedure. The monitoring mayalso include the detection of the expression of additional genes orgenetic elements, e.g. housekeeping genes like GAPDH or PBGD, or otherphosphodiesterases, preferably PDE4D5.

The term “prognosticating prostate cancer” as used herein refers to theprediction of the course or outcome of a diagnosed or detected cancerousdisease, e.g. during a certain period of time, during a treatment orafter a treatment. The term also refers to a determination of chance ofsurvival or recovery from a disease, as well as to a prediction of theexpected survival time of a subject. A prognosis may, specifically,involve establishing the likelihood for survival of a subject during aperiod of time into the future, such as 6 months, 1 year, 2 years, 3years, 5 years, 10 years or any other period of time.

The term “progression of prostate cancer” as used herein relates to aswitch between different stages of prostate cancer development, e.g.stages 0 and I to IV of the TNM classification, or any other stage orsub-stage, starting from a healthy condition up to a terminal cancerscenario. Typically such switches are accompanied by a modification ofthe expression level of PDE4D7, preferably a decrease, in a test samplein comparison to a previous test sample from the same individual, e.g.in comparison to a sample derived from a hormone-dependent prostatetumor or tumor control or a hormone-sensitive prostate tumor or tumorcontrol. A progression of prostate cancer may be considered as beingdetected or diagnosed if the PDE4D7 expression level, as defined hereinabove, is decreased by a value of between 3% to 50%, preferably by avalue of 10%, 15%, 20% or 25% in a test sample in comparison to aprevious test sample from the same individual. The modification may bedetected over any period of time, preferably over 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12 months, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20years, i.e. the value indicated above may be calculated by comparing theexpression level of PDE4D7 at a first point in time and at a secondpoint in time after the above indicated period of time. The progressionmay, in a specific embodiment, be a progression towardshormone-resistant prostate cancer.

In a particularly preferred embodiment of the present invention the term“progression of prostate cancer” relates to a switch from ahormone-dependent or hormone-sensitive prostate cancer state to ahormone-resistant prostate cancer state.

A progression from a hormone-dependent or hormone-sensitive prostatecancer state to a hormone-resistant prostate cancer state may beconsidered as being detected or diagnosed if the PDE4D7 expressionlevel, as defined herein above, is decreased by a value of between 3% to50%, preferably by a value of 10%, 15%, 20% or 25% in a test sample incomparison to a previous test sample from the same individual, which hasbeen diagnosed as suffering from a hormone-sensitive orhormone-dependent prostate cancer. The progression may also beconsidered to be detected if the comparison is carried out with testsamples from other individuals, test samples from tissue collections,values from databases etc.

The modification may be detected over any period of time, preferablyover 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, 1.5, 2, 3, 4, 5, 6,7, 8, 9, 10, 15 or 20 years, i.e. the value indicated above may becalculated by comparing the expression level of PDE4D7 at a first pointin time and at a second point in time after the above indicated periodof time.

In a further embodiment the present invention relates to the diagnosisand detection of a predisposition for developing prostate cancer, morepreferably hormone-resistant prostate cancer. A “predisposition fordeveloping prostate cancer” and in particular a “predisposition fordeveloping hormone-resistant prostate cancer” in the context of thepresent invention is a state of risk of developing prostate cancer, inparticular hormone-resistant prostate cancer. Preferably apredisposition for developing hormone-resistant prostate cancer may bepresent in cases in which the PDE4D7 expression level as defined hereinabove is below a cancerous control level as defined herein above, e.g. areference expression level derived from tissues or samples of a subjectwhich evidently suffers from hormone-sensitive prostate cancer. The term“below” as used herein means that the expression level of PDE4D7 isdecreased by about 40% to 80% in comparison to a cancerous controllevel, preferably decreased by about 50%.

Alternatively, a predisposition for developing cancer in the context ofthe present invention may be present in situations in which the PDE4D7expression level as defined herein above is given and in which further,alternative cancer markers, e.g. PSA, show no modification of expressionlevel or the expression pattern. Suitable further cancer markers areknown to the person skilled in the art.

Thus, a predisposition for prostate cancer, in particularhormone-resistant prostate cancer, may be considered as being diagnosedor detected if one of the above depicted situations is observed.

The difference between the expression levels of a test biological sampleand a control level can be normalized to the expression level of furthercontrol nucleic acids, e.g. housekeeping genes whose expression levelsare known not to differ depending on the cancerous or non-cancerousstate of the cell. Exemplary control genes include inter alia β-actin,glycerinaldehyde 3-phosphate dehydrogenase (GAPDH), porphobilinogendeanimase (PBGD) and ribosomal protein P1. The normalization may also becarried out with other phosphodiesterases, preferably with a humanphosphodiesterase showing an unaltered expression pattern in differenttumor stages. A preferred phosphodiesterase is PDE4D5 or any otherisoform of the PDE4D family, e.g. PDE4D1, PDE4D2, PDE4D3, PDE4D4,PDE4D6, PDE4D8 or PDE4D9.

In the context of the present invention, the terms “diagnosing” and“prognosticating” are also intended to encompass predictions andlikelihood analyses. PDE4D7 as a marker may accordingly be usedclinically in making decisions concerning treatment modalities,including therapeutic intervention or diagnostic criteria such as asurveillance for the disease. According to the present invention, anintermediate result for examining the condition of a subject may beprovided. Such intermediate result may be combined with additionalinformation to assist a doctor, nurse, or other practitioner to diagnosethat a subject suffers from the disease. Alternatively, the presentinvention may be used to detect cancerous cells in a subject-derivedtissue, and provide a doctor with useful information to diagnose thatthe subject suffers from the disease.

A subject or individual to be diagnosed, monitored or in which aprostate cancer, a progression of prostate cancer or predisposition forprostate cancer is to be detected or prognosticated according to thepresent invention is an animal, preferably a mammal, more preferably ahuman being.

Particularly preferred is the use of molecular imaging tools as known tothe person skilled in the art, e.g. magnetic resonance imaging (MRI)and/or magnetic photon resonance imaging (MPI) technology in the contextof using PDE4D7 as a marker for diagnosing, detecting, monitoring orprognosticating prostate cancer of the progression of prostate cancer.For example, PDE4D7 may be used as a marker for diagnosing, detecting,monitoring or prognosticating prostate cancer or the progression ofprostate cancer in approaches like MRI or MPI that allows for onlinedetection of the diagnostic marker within a human subject.

In a further aspect the present invention relates to a composition fordiagnosing, detecting, monitoring or prognosticating prostate cancer orthe progression of prostate cancer or a predisposition for prostatecancer in an individual. The composition according to the presentinvention may comprise a nucleic acid or peptide affinity ligand for thePDE4D7 expression product or protein.

The term “nucleic acid affinity ligand for the PDE4D7 expressionproduct” as used herein refers to a nucleic acid molecule being able tospecifically bind to a PDE4D7 transcript or a DNA molecule derived fromsplice variant 7 of PDE4D, even more preferably to the DNA sequencedepicted in SEQ ID NO: 1 or to the complementary DNA sequence of thesequence depicted in SEQ ID NO: 1 or a corresponding RNA molecule. Thenucleic acid affinity ligand may also be able to specifically bind to aDNA sequence being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ IDNO: 1 or a DNA sequence encoding an amino acid sequence being at least75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to the sequence as set forth in SEQ ID NO: 2 or to anyfragments of said sequences.

The term “peptide affinity ligand for the PDE4D7 protein” as used hereinrefers to a peptide molecule being able to specifically bind to thePDE4D7 protein. The peptide molecule may preferably be able tospecifically bind to a protein or polypeptide comprising the amino acidsequence as set forth in SEQ ID NO: 2. The peptide affinity ligand mayalso be able to specifically bind to a protein or polypeptide comprisingan amino acid sequence encoded by a DNA sequence being at least 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identicalto the sequence as set forth in SEQ ID NO: 1 or to a protein orpolypeptide comprising an amino acid sequence being at least 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to thesequence as set forth in SEQ ID NO: 2 or to any fragments of saidsequences. The term “peptide” refers to any type of amino acid sequencecomprising more than 2 amino acids, e.g. polypeptide structures, proteinstructures or functional derivatives thereof. Furthermore, the peptidemay be combined with further chemical moieties or functionalities.

The term “expression product” as used herein refers to a PDE4D7transcript or an mRNA molecule generated by the expression of the PDE4Dgene. More preferably, the term relates to a processed PDE4D transcriptas defined herein above, e.g. via the sequence as set forth in SEQ IDNO: 1.

In a preferred embodiment of the present invention the composition ofthe present invention comprises nucleic acid and peptide affinityligands selected from the group consisting of a set of oligonucleotidesspecific for the PDE4D7 expression product, a probe specific for thePDE4D7 expression product, an aptamer specific for the PDE4D7 expressionproduct or for the PDE4D7 protein, an antibody specific for the PDE4D7protein and an antibody variant specific for the PDE4D7 protein.

The composition of the present invention may, for example, comprise aset of oligonucleotides specific for the PDE4D7 expression productand/or a probe specific for the PDE4D7 expression product. The term“oligonucleotide specific for the PDE4D7 expression product” as usedherein refers to a nucleotide sequence which is complementary to thesense- or antisense-strand of splice variant 7 of PDE4D. Preferably, theoligonucleotide is complementary to the DNA sequence depicted in SEQ IDNO: 1 or to the complementary DNA sequence of the sequence depicted inSEQ ID NO: 1. The oligonucleotide sequence may also be complementary toa DNA sequence being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQID NO: 1 or a DNA sequence encoding an amino acid sequence being atleast 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to the sequence as set forth in SEQ ID NO: 2.

The oligonucleotide may have any suitable length and sequence known tothe person skilled in the art, as derivable from the sequence of SEQ IDNO: 1 or its complement. Typically, the oligonucleotide may have alength of between 8 and 60 nucleotides, preferably of between 10 and 35nucleotides, more preferably a length of 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33nucleotides. Oligonucleotide sequences specific for the PDE4D7expression product may be defined with the help of software tools knownto the person skilled in the art.

In a further embodiment of the present invention the oligonucleotidesequences may be complementary to sequences localized in exon 1 or exon2 of the PDE4D7 gene, to sequences localized in the boundary betweenexon 1 and exon 2 of the PDE4D7 gene or to sequences localized in exon 2of the PDE4D7 gene solely, preferably to a stretch of 271 uniquenucleotides of PDE4D7, i.e. 42 nucleotides at the 3′ end of exon 1 and229 5′-terminal nucleotides of exon 2 of PDE4D. For instance, anoligonucleotide usable as a forward primer may be localized at theboundary between exon 1 and exon 2 of the PDE4D7 gene and theoligonucleotide usable as a reverse primer may be localized in exon 2 ofthe PDE4D7 gene.

In a preferred embodiment of the present invention the set ofoligonucleotides has the sequences as set forth in SEQ ID NO: 3 and SEQID NO: 4. Further preferred are the oligonucleotides having orcomprising the sequence as set forth in SEQ ID NO: 7 and/or SEQ ID NO:8.

The term “probe specific for the PDE4D7 expression product” as usedherein means a nucleotide sequence which is complementary to the sense-or antisense-strand of splice variant 7 of PDE4D. Preferably, the probeis complementary to the DNA sequence depicted in SEQ ID NO: 1 or to thecomplementary DNA sequence of the sequence depicted in SEQ ID NO: 1. Theprobe sequence may also be complementary to a DNA sequence being atleast 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to the sequence as set forth in SEQ ID NO: 1 or a DNA sequenceencoding an amino acid sequence being at least 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence asset forth in SEQ ID NO: 2.

The probe may have any suitable length and sequence known to the personskilled in the, as derivable from the sequence of SEQ ID NO: 1 or itscomplement. Typically, the probe may have a length of between 6 and 300nucleotides, preferably of between 15 and 60 nucleotides, morepreferably a length of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49or 50 nucleotides. Probe sequences specific for the PDE4D7 expressionproduct may be defined with the help of software tools known to theperson skilled in the art.

In a further embodiment of the present invention the probe sequence maybe complementary to a sequence localized in exon 1 or exon 2 of thePDE4D7 gene, preferably to a stretch of 271 unique nucleotides ofPDE4D7, i.e. 42 nucleotides at the 3′ end of exon 1 and 229 5′-terminalnucleotides of exon 2 of PDE4D. If the probe is to be used forquantitative PCR reactions, e.g. real time PCR, the probe may bedesigned such that it is localized at a position in between the bindingpositions of a forward and reverse primer. Preferably, the probe may bedesigned such that it is localized in the proximity of one of the primeroligonucleotides. More preferably, it may be localized in the proximityof the forward primer.

In a preferred embodiment of the present invention the probe has thesequence as set forth in SEQ ID NO: 5 or SEQ ID NO: 9.

The composition of the present invention may additionally oralternatively comprise an aptamer specific for the PDE4D7 expressionproduct or protein. The term “aptamer specific for the PDE4D7 expressionproduct” as used herein refers to a short nucleic acid molecule, e.g.RNA, DNA, PNA, CNA, HNA, LNA or ANA or any other suitable nucleic acidformat known to the person skilled in the art, being capable ofspecifically binding to splice variant 7 of PDE4D, preferably the DNAmolecule derived from splice variant 7 of PDE4D. More preferably, thenucleic acid aptamer molecule may specifically bind to a DNA sequencedepicted in SEQ ID NO: 1 or a double stranded derivative thereof. Thenucleic acid aptamer according to the present invention may also bind toan RNA molecule corresponding to the PDE4D7 transcript, preferably anRNA molecule corresponding to the DNA sequence as set forth in SEQ IDNO: 1.

The nucleic acid aptamer may further be capable of specifically bindingto a DNA sequence being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQID NO: 1 or a DNA sequence encoding an amino acid sequence being atleast 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to the sequence as set forth in SEQ ID NO: 2 or RNA moleculescorresponding to these sequences.

Specificity of the nucleic acid aptamer to splice variant 7 of PDE4D maybe conferred by a specific binding to sequences solely present in saidsplice variant, e.g. exon 2 or the exon boundary between exon 1 and exon2 of PDE4D. In a particular embodiment of the present inventionspecificity of the nucleic acid aptamer to splice variant 7 of PDE4D maybe conferred by a specific binding to a sequence located within astretch of 271 unique nucleotides of PDE4D7, i.e. 42 nucleotides at the3′ end of exon 1 and 229 5′-terminal nucleotides of exon 2 of PDE4D.Nucleic acid aptamers may be generated according to any suitable methodknown to the person skilled in the art, e.g. by in vitro selection orSELEX methods. Preferably, nucleic acid aptamers may be generated and/ordesigned according to the guidance provided in Ellington and Szostak,1990, Nature, 346:818-822. A nucleic acid aptamer according to thepresent invention may further be combined with additional moieties, e.g.with interacting portions like biotin or enzymatic functionalities likeribozyme elements.

The term “aptamer specific for the PDE4D7 protein” as used herein refersto a short peptide capable of interacting and specifically binding thePDE4D7 protein. The peptide aptamer may preferably be able tospecifically bind to a protein or polypeptide comprising the amino acidsequence as set forth in SEQ ID NO: 2. The peptide aptamer may also beable to specifically bind to a protein or polypeptide comprising anamino acid sequence encoded by a DNA sequence being at least 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to thesequence as set forth in SEQ ID NO: 1 or to a protein or polypeptidecomprising an amino acid sequence being at least 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequenceas set forth in SEQ ID NO: 2. Typically, a peptide aptamer is a variablepeptide loop, comprising for example, 10 to 20 amino acids. In thecontext of the present invention the peptide aptamer may preferably beattached at one or both ends to a scaffold structure. The scaffoldstructure may be any molecule, preferably a protein, which has goodsolubility properties. Suitable scaffold molecules would be known to theperson skilled in the art. A preferred scaffold molecule to be used inthe context of the present invention is the bacterial proteinthioredoxin-A. The aptamer peptide loop may preferably be insertedwithin a reducing active site of the scaffold molecule. Alternatively,staphylococcal protein A and domains thereof and derivatives of thesedomains, such as protein Z or lipocalins may be used as scaffoldstructures in the context of the present invention.

Peptide aptamers may be generated according to any suitable method knownto the person skilled in the art, e.g. via yeast two-hybrid approaches.

In another preferred embodiment of the present invention the compositionmay comprise, or may additionally comprise, an antibody specific for thePDE4D7 protein, preferably a monoclonal or polyclonal antibody. Alsopreferred are antibody variants or fragments like a single chainantibody, a diabody, a minibody, a single chain Fv fragment (sc(Fv)), asc(Fv)₂ antibody, a Fab fragment or a F(ab′)₂ fragment based on amonoclonal PDE4D7 specific antibody, a small modularimmunopharmaceutical (SMIP), a binding-domain immunoglobulin fusionprotein, a camelized antibody, a V_(HH) containing antibody etc. Theantibody may be mono-, bi-, tri- or multivalent. The antibody may be ofany origin, e.g. a murine, human, or chimeric, or a humanized murineantibody. In a specific embodiment of the present invention commerciallyavailable anti-PDE4D7 antibodies like NB300-652 (Novus Biologicals,Inc.) or GTX14629 (GeneTex, Inc.) may be comprised in the composition ormay be used diagnostically.

Antibodies may be produced according to any suitable method known to theperson skilled in the art. Polyclonal antibodies may be produced byimmunization of animals with the antigen of choice, whereas monoclonalantibodies of defined specificity may be produced using, for instance,the hybridoma technology developed by Köhler and Milstein (Köhler andMilstein, 1976, Eur. J. Immunol., 6:511-519).

An affinity ligand, as described herein above, may be labeled withvarious markers or may be detected by a secondary affinity ligand,labeled with various markers, to allow detection, visualization and/orquantification. This can be accomplished using any suitable labels,which can be conjugated to the affinity ligand capable of interactionwith the PDE4D7 expression product or the PDE4D7 protein or to anysecondary affinity ligand, using any suitable technique or methods knownto the person skilled in the art. The term “secondary affinity ligand”refers to a molecule which is capable of binding to the affinity ligandas defined herein above (i.e. a “primary affinity ligand” if used in thecontext of a system with two interacting affinity ligands). The bindinginteraction is preferably a specific binding.

Examples of labels that can be conjugated to a primary and/or secondaryaffinity ligands include fluorescent dyes or metals (e.g. fluorescein,rhodamine, phycoerythrin, fluorescamine), chromophoric dyes (e.g.rhodopsin), chemiluminescent compounds (e.g. luminal, imidazole) andbioluminescent proteins (e.g. luciferin, luciferase), haptens (e.g.biotin).

In a particularly preferred embodiment an affinity ligand to be used asa probe, in particular a probe specific for the PDE4D7 expressionproduct as defined herein above, may be labeled with a fluorescent labellike 6-FAM, HEX, TET, ROX, Cy3, Cy5, Texas Red or Rhodamine, and/or atthe same time with a quenching label like TAMRA, Dabcyl, Black HoleQuencher, BHQ-1 or BHQ-2. A variety of other useful fluorescents andchromophores are described in Stryer, 1968, Science, 162:526-533.Affinity ligands may also be labeled with enzymes (e.g. horseradishperoxidase, alkaline phosphatase, beta-lactamase), radioisotopes (e.g.³H, ¹⁴C, ³²P, ³³P, ³⁵S, ¹²⁵I, ¹¹C, ¹³N, ¹⁵O, ¹⁸F, ⁶⁴Cu, ⁶²Cu, ¹²⁴I,⁷⁶Br, ⁸²Rb, ⁶⁸Ga or ¹⁸F) or particles (e.g. gold).

The different types of labels may be conjugated to an affinity ligandusing various chemistries, e.g. the amine reaction or the thiolreaction. However, other reactive groups than amines and thiols can alsobe used, e.g. aldehydes, carboxylic acids and glutamine.

In a preferred embodiment of the present invention the nucleic acidaffinity ligand or peptide affinity ligand of the present invention maybe modified to function as a contrast agent.

The term “contrast agent” as used herein refers to a molecular compoundthat is capable of specifically interacting with the PDE4D7 marker andwhich can be detected by an apparatus positioned outside the human oranimal body. Preferably, such contrast agents are suitable for use inmagnetic resonance imaging (MRI) or magnetic photon imaging (MPI). Theterm “specifically interacting” refer to the property of a molecularcompound to preferentially interact with the PDE4D7 marker on the cellsurface of cells being present within the human or animal body overother proteins that are expressed by such cells. Preferred contrastagents which may also be designated as contrast agent compositions willbe capable of specifically detecting molecules having the nucleotidesequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 2 orderivatives or homologous variants thereof as defined herein above.Preferred contrast agents are aptamers specific for the PDE4D7expression product or for the PDE4D7 protein as defined herein above aswell as antibodies specific for the PDE4D7 protein as defined hereinabove.

Contrast agents, aside from their property of being capable ofspecifically recognizing the PDE4D7 marker will in addition typicallycomprise a further molecule which is detectable by the specificdetection technology used. The term “modified to function” as usedherein thus refers to any suitable modifications known to the personskilled in the art, which may be necessary in order to allow the use ofthe contrast agent in molecular imaging methods, in particular in MRI orMPI. For example, if fluorescent spectroscopy is used as a detectionmeans, such molecules may comprise fluorophores as detectable markermolecules that can be excited at a specific wavelength. Alternatively, aradioactive label, e.g. a radioisotope as described herein above may beemployed. With respect to preferred contrast agents in accordance withthe invention that are suitable for MRI, the contrast agents such as theabove described antibodies may comprise a marker molecule which isdetectable by MRI. Such detectable labels include e.g. USPIOS and19Fluor.

In a specific embodiment of the present invention a composition mayadditionally comprise accessory ingredients like PCR buffers, dNTPs, apolymerase, ions like bivalent cations or monovalent cations,hybridization solutions, secondary affinity ligands like, e.g. secondaryantibodies, detection dyes and any other suitable compound or liquidnecessary for the performance of a detection based on any of theaffinity ligands or contrast agents as defined herein above, which isknown to the person skilled in the art.

In another aspect the present invention relates to the use of a nucleicacid or peptide affinity ligand for the PDE4D7 expression product orprotein, as defined herein above, for the preparation of a compositionfor diagnosing, detecting, monitoring or prognosticating prostate canceror the progression of prostate cancer or a predisposition for prostatecancer in an individual, as described herein above.

In a preferred embodiment the present invention relates to the use of aset of oligonucleotides specific for the PDE4D7 expression productand/or a probe specific for the PDE4D7 expression product, as definedherein above, for the preparation of a composition for diagnosing,detecting, monitoring or prognosticating prostate cancer or theprogression of prostate cancer or a predisposition for prostate cancerin an individual, as described herein above. In another preferredembodiment the present invention relates to the use of an aptamerspecific for the PDE4D7 expression product or protein, as defined hereinabove, for the preparation of a composition for diagnosing, detecting,monitoring or prognosticating prostate cancer or the progression ofprostate cancer or a predisposition for prostate cancer in anindividual, as described herein above.

In a further preferred embodiment the present invention relates to theuse of an antibody specific for the PDE4D7 protein or an antibodyvariant specific for the PDE4D7 protein, as defined herein above, forthe preparation of a composition for diagnosing, detecting, monitoringor prognosticating prostate cancer or the progression of prostate canceror a predisposition for prostate cancer in an individual, as describedherein above.

In a preferred embodiment of the present invention a composition asdefined herein above is a diagnostic composition.

In another aspect the present invention relates to a diagnostic kit fordetecting, diagnosing, monitoring or prognosticating prostate cancer orthe progression of prostate cancer or a predisposition for prostatecancer, comprising a set of oligonucleotides specific for the PDE4D7expression product, a probe specific for the PDE4D7 expression productand/or an aptamer specific for the PDE4D7 expression product or proteinand/or an antibody specific for the PDE4D7 protein and an antibodyvariant specific for the PDE4D7 protein.

Typically, the diagnostic kit of the present invention contains one ormore agents allowing the specific detection of PDE4D7 as defined hereinabove. The agents or ingredients of a diagnostic kit may, according tothe present invention, be comprised in one or more containers orseparate entities. The nature of the agents is determined by the methodof detection for which the kit is intended. Where detection at thePDE4D7 mRNA expression level, i.e. via the PDE4D7 expression product, isintended, the agents to be comprised may be a set of oligonucleotidesspecific for the PDE4D7 expression product and/or a probe specific forthe PDE4D7 expression product as defined herein above, which may beoptionally labeled according to methods known in the art, e.g. withlabels described herein above. In addition or alternatively an aptamerspecific for the PDE4D7 expression production may be comprised. Wheredetection is at the PDE4D7 protein level is intended, the agents to becomprised may be antibodies or compounds containing an antigen-bindingfragment of an antibody or antibody variants specific for the PDE4D7protein, as described herein above. In addition or alternatively anaptamer specific for the PDE4D7 protein may be comprised. Alternatively,a diagnostic kit may comprise a contrast agent as defined herein above.

The presence of specific proteins may also be detected using othercompounds that specifically interact with PDE4D7, e.g. specificsubstrates or ligands.

Preferably, a diagnostic kit of the present invention contains detectionreagents for PDE4D7 expression product or the PDE4D7 protein. Suchdetection reagents comprise, for example, buffer solutions, labels orwashing liquids etc. Furthermore, the kit may comprise an amount of aknown nucleic acid molecule or protein, which can be used for acalibration of the kit or as an internal control. Typically, adiagnostic kit for the detection of PDE4D7 expression products maycomprise accessory ingredients like a PCR buffers, dNTPs, a polymerase,ions like bivalent cations or monovalent cations, hybridizationsolutions etc. A diagnostic kit for the detection of PDE4D7 proteins mayalso comprise accessory ingredients like secondary affinity ligands,e.g. secondary antibodies, detection dyes and any other suitablecompound or liquid necessary for the performance of a protein detectionbased known to the person skilled in the art. Such ingredients are knownto the person skilled in the art and may vary depending on the detectionmethod carried out. Additionally, the kit may comprise an instructionleaflet and/or may provide information as to the relevance of theobtained results.

In another aspect the present invention relates to a method fordetecting, diagnosing, monitoring or prognosticating prostate cancer orthe progression of prostate cancer in an individual comprising at leastthe step of determining the level of PDE4D7 in a sample. The term“determining the level of PDE4D7” refers to the determination of thepresence or amount of PDE4D7 expression products, e.g. PDE4D7transcript(s), and/or the determination of the presence and/or amount ofPDE4D7 protein(s). The term “level of PDE4D7” thus means the presence oramount of PDE4D7 expression products, e.g. PDE4D7 transcript(s), and/orthe determination of the presence or amount of PDE4D7 protein(s). Thedetermination of the presence or amount of PDE4D7 expression products,e.g. PDE4D7 transcript(s) or PDE4D7 protein(s) may be accomplished byany means known in the art.

In a preferred embodiment of the present invention the determination ofthe presence or amount of PDE4D7 expression products, e.g. PDE4D7transcript(s) and/or of PDE4D7 protein(s), is accomplished by themeasurement of nucleic acid or protein levels or by the determination ofthe biological activity of PDE4D7. Thus, the PDE4D7 expression level(s)may be determined by a method involving the detection of an mRNA encodedby the PDE4D7 gene, the detection of the PDE4D7 protein encoded by thePDE4D7 transcript and/or the detection of the biological activity of thePDE4D7 protein.

For example, the measurement of the nucleic acid level of PDE4D7expression may be assessed by separation of nucleic acid molecules (e.g.RNA or cDNA) obtained from the sample in agarose or polyacrylamide gels,followed by hybridization with PDE4D7 specific oligonucleotide probes asdefined herein above. Alternatively, the expression level may bedetermined by the labeling of nucleic acid obtained from the samplefollowed by separation on a sequencing gel. Nucleic acid samples may beplaced on the gel such that patient and control or standard nucleic acidare in adjacent lanes. Comparison of expression levels may beaccomplished visually or by means of a densitometer. Methods for thedetection of mRNA or expression products are known to the person skilledin the art. Typically, Northern blot analysis may be used for such apurpose.

Alternatively, the nucleic acid level of PDE4D7 expression may bedetected in a DNA array or microarray approach. Typically, samplenucleic acids derived from subjects to be tested are processed andlabeled, preferably with a fluorescent label. Subsequently, such nucleicacid molecules may be used in a hybridization approach with immobilizedcapture probes corresponding to the PDE4D7 marker gene of the presentinvention or known biomarker or cancer marker genes. Suitable means forcarrying out microarray analyses are known to the person skilled in theart.

In a standard setup a DNA array or microarray comprises immobilizedhigh-density probes to detect a number of genes. The probes on the arrayare complementary to one or more parts of the sequence of the markergene, or to the entire coding region of the marker gene. In the presentinvention, any type of PDE4D7 associated polynucleotide may be used asprobe for the DNA array, as long as the polynucleotide allows for aspecific distinction between PDE4D7 expression and the expression ofother genes. Typically, cDNAs, PCR products, and oligonucleotides areuseful as probes. Preferably, a probe involving the specific portions ofsplice variant 7 of PDE4D may be used as a probe. In addition to thedetermination of the PDE4D7 expression also the determination of theexpression of other genes, e.g. additional biomarker or cancer markergenes may be accomplished.

A DNA array- or microarray-based detection method typically comprisesthe following steps: (1) Isolating mRNA from a sample and optionallyconverting the mRNA to cDNA, and subsequently labeling this RNA or cDNA.Methods for isolating RNA, converting it into cDNA and for labelingnucleic acids are described in manuals for micro array technology. (2)Hybridizing the nucleic acids from step 1 with probes for the markergenes. The nucleic acids from a sample can be labeled with a dye, suchas the fluorescent dyes Cy3 (red) or Cy5 (blue). Generally a controlsample is labeled with a different dye. (3) Detecting the hybridizationof the nucleic acids from the sample with the probes and determining atleast qualitatively, and more particularly quantitatively, the amountsof mRNA in the sample for PDE4D7 and/or additional marker genesinvestigated. The difference in the expression level between sample andcontrol can be estimated based on a difference in the signal intensity.These can be measured and analyzed by appropriate software such as, butnot limited to the software provided for example by Affymetrix.

There is no limitation on the number of probes corresponding to themarker genes used, which are spotted on a DNA array. Also, a marker genecan be represented by two or more probes, the probes hybridizing todifferent parts of a gene. Probes are designed for each selected markergene. Such a probe is typically an oligonucleotide comprising 5-50nucleotide residues. Longer DNAs can be synthesized by PCR orchemically. Methods for synthesizing such oligonucleotides and applyingthem on a substrate are well known in the field of micro-arrays. Genesother than the marker genes may be also spotted on the DNA array. Forexample, a probe for a gene whose expression level is not significantlyaltered may be spotted on the DNA array to normalize assay results or tocompare assay results of multiple arrays or different assays.

Alternatively, the nucleic acid level of PDE4D7 expression may bedetected in a quantitative RT-PCR approach, preferably in a real-timePCR approach following the reverse transcription of the PDE4D7 mRNAtranscript. Typically, as first step, a transcript is reversetranscribed into a cDNA molecule according to any suitable method knownto the person skilled in the art. A quantitative or real-time PCRapproach may subsequently be carried out based on a first DNA strandobtained as described above.

Preferably, Taqman or Molecular Beacon probes as principal FRET-basedprobes of this type may be used for quantitative PCR detection. In bothcases, the probes, preferably PDE4D7 probes as defined herein above,serve as internal probes which are used in conjunction with a pair ofopposing primers that flank the target region of interest, preferably aset of PDE4D7 oligonucleotides as defined herein above. Uponamplification of a target segment, the probe may selectively bind to theproducts at an identifying sequence in between the primer sites, therebycausing increases in FRET signaling relative to increases in targetfrequency.

Preferably, a Taqman probe to be used for a quantitative PCR approachaccording to the present invention may comprises a PDE4D7oligonucleotide as defined above of about 22 to 30 bases that is labeledon both ends with a FRET pair. Typically, the 5′ end will have a shorterwavelength fluorophore such as fluorescein (e.g. FAM) and the 3′ end iscommonly labeled with a longer wavelength fluorescent quencher (e.g.TAMRA) or a non-fluorescent quencher compound (e.g. Black HoleQuencher). It is preferred that the probes to be used for quantitativePCR, in particular the PDE4D7 probes as defined herein above, have noguanine (G) at the 5′ end adjacent to the reporter dye in order to avoidquenching of the reporter fluorescence after the probe is degraded.

A Molecular Beacon probe to be used for a quantitative PCR approachaccording to the present invention preferably uses FRET interactions todetect and quantify a PCR product, with each probe having a 5′fluorescent-labeled end and a 3′ quencher-labeled end. This hairpin orstem-loop configuration of the probe structure comprises preferably astem with two short self-binding ends and a loop with a long internaltarget-specific region of about 20 to 30 bases.

Alternative detection mechanisms which may also be employed in thecontext of the present invention are directed to a probe fabricated withonly a loop structure and without a short complementary stem region. Analternative FRET-based approach for quantitative PCR which may also beused in the context of the present invention is based on the use of twohybridization probes that bind to adjacent sites on the target whereinthe first probe has a fluorescent donor label at the 3′ end and thesecond probe has a fluorescent acceptor label at its 5′ end.

The measurement of protein levels of the PDE4D7 protein or of anyfragments, homologues or derivates derived thereof may be carried outvia any suitable detection technique known in the art. Preferably, theprotein level of PDE4D7 and derivatives thereof may be determinedimmunologically, e.g. by using an antibody specific for the PDE4D7protein, preferably an antibody as defined herein above. Alternatively,antibody variants or fragments as defined herein above may be used. Thepresent invention also envisages the use of peptide affinity ligandslike aptamers specific for the PDE4D7 protein as defined herein above.

Determination of the protein levels of the PDE4D7 protein can beaccomplished, for example, by the separation of proteins from a sampleon a polyacrylamide gel, followed by identification of the PDE4D7protein using specifically binding antibodies in a Western blotanalysis. Alternatively, proteins can be separated by two-dimensionalgel electrophoresis systems. Two-dimensional gel electrophoresis is wellknown in the art and typically involves iso-electric focusing along afirst dimension followed by SDS-PAGE electrophoresis along a seconddimension. The analysis of 2D SDS-PAGE gels can be performed bydetermining the intensity of protein spots on the gel, or can beperformed using immune detection. In other embodiments, protein samplesare analyzed by mass spectroscopy.

Within the context of the present invention PDE4D7 specific antibodiesmay be placed on a support and be immobilized. Proteins derived fromsamples or tissues to be analyzed may subsequently be mixed with theantibodies. A detection reaction may then be carried out, e.g. with asecond affinity ligand as defined herein above, preferably with aspecific antibody.

Immunological tests which may be used in the context of the presentinvention, in particular for the diagnostic purposes of the presentinvention, include, for example, competitive and non-competitive assaysystems using techniques such as western blots, radioimmunoassay likeRIA (radio-linked immunoassay), ELISA (enzyme linked immunosorbentassay), “sandwich” immunoassays, immunoprecipitation assays, precipitinreactions, gel diffusion precipitin reactions, immunodiffusion assays,agglutination assays, e.g. latex agglutination, complement-fixationassays, immunoradiometric assays, fluorescent immunoassays, e.g. FIA(fluorescence-linked immunoassay), chemiluminescence immunoassays,electrochemiluminescence immunoassay (ECLIA) and protein A immunoassays.Such assays are routine and well known to the person skilled in the art.

Furthermore, the binding affinity of an antibody to an antigen and theoff-rate of an antibody-antigen interaction may be determined bycompetitive binding assays. One example of a competitive binding assayis a radioimmunoassay comprising the incubation of labeled antigen(e.g., ³H or ¹²⁵I) with a suitable antibody in the presence ofincreasing amounts of unlabeled antigen, and the detection of theantibody bound to the labeled antigen. The affinity of the antibody ofinterest for a particular antigen and the binding off-rates may bedetermined from the data by any suitable analysis approach, e.g. by ascatchard plot analysis. Competition with a second antibody may also bedetermined using radioimmunoassays. In this case, the antigen may beincubated with a suitable antibody conjugated to a labeled compound(e.g., ³H or ¹²⁵I) in the presence of increasing amounts of an unlabeledsecond antibody.

In addition, aptamers specific for the PDE4D7 protein, preferably asdefined herein above, may be used in a method of detecting PDE4D7proteins. Such aptamers may preferably be labeled in order to allow thedetection of a protein-ligand interaction.

The determination of the biological activity of PDE4D7 may be carriedout by employing molecular or enzymatic assays specific to thecorresponding function or functions of PDE4D7. Preferably, a readoutsystem based on the conversion of cAMP by phosphodiesterase may be used.Suitable techniques would be known to the person skilled in the art. Ina further preferred embodiment, an assay for the determination of thebiological activity of PDE4D7 may be carried out in combination with theinhibition of the activity of other PDE4D splice variants, other PDE4iso forms and/or other PDEs, preferably other PDEs capable of performingthe conversion of cAMP. Such an inhibition of the activity may becarried out by any suitable means known to the person skilled in theart, preferably via the use of suitable antisense nucleotides, siRNAmolecules or miRNA molecules, more preferably via specificallyhybridizing antisense nucleotides, specific siRNA or miRNA molecules.

In a further preferred embodiment the biological activity of PDE4D7 maybe tested with the help of specific PDE4D7 inhibitors. The use of suchinhibitors may, for example, be combined with a readout system based onthe conversion of the cAMP substrate. Typical PDE4D7 inhibitors to beused comprise antisense molecules, siRNA molecules or miRNA molecules.

The level of PDE4D7 may also be detected in methods involvinghistological or cell-biological procedures. Typically, visualtechniques, such as light microscopy or immunofluoresence microscopy, aswell as flow cytometry or luminometry may be used. The presence ofPDE4D7 protein in a cell may, for instance, be detected or determined byremoving cells to be tested from samples as defined herein above. Alsotissue sections or biopsy samples may be used for these methods.Subsequently, affinity ligands for PDE4D7 may be applied, preferablyantibodies or aptamers. Typically, such affinity ligands are labeled,preferably with fluorescent labels as defined herein above. Such aprocedure allows for the detection of PDE4D7, for its quantificationand, in addition, allows to determine the distribution and relativelevel of expression thereof.

Such procedures involve the use of visualization methods. Suitablevisualization methods are known to the person skilled in the art.Typical methods to be used comprise fluorometric, luminometric and/orenzymatic techniques. Fluorescence is normally detected and/orquantified by exposing fluorescent labels to light of a specificwavelength and thereafter detecting and/or quantifying the emitted lightof a specific wavelength. The presence of a luminescently taggedaffinity ligand may be detected and/or quantified by luminescencedeveloped during a chemical reaction. Detection of an enzymatic reactionis due to a color shift in the sample arising from chemical reaction.

In a further, preferred embodiment the level of PDE4D7 may be determinedby suitable molecular imaging techniques, e.g. magnetic resonanceimaging (MRI) or magnetic photon imaging (MPI), and/or by using suitablecontrast agents, e.g. contrast agents as defined herein above.

In a further, preferred embodiment a method for detecting, diagnosing,monitoring or prognosticating prostate cancer or the progression ofprostate cancer of the present invention comprises the additional stepof comparing the measured nucleic acid or protein levels or the measuredbiological activity to a control level. The term “control level” as usedherein refers to the expression of the PDE4D7 marker or other suitablemarkers in a cancerous control or non-cancerous control, as definedherein above. The status, nature, amount and condition of the controllevel may be adjusted according to the necessities. Preferably anon-cancerous control level may be used. The term “comparing” as usedherein refers to any suitable method of assessing, calculating,evaluating or processing of data.

In yet another embodiment as a further, additional step a decision onthe presence or stage of prostate cancer or the progression of prostatecancer may be based on the results of the comparison step. A prostatecancer may be diagnosed or prognosticated or a progression of prostatecancer may be diagnosed or prognosticated in said method according tothe corresponding definitions provided herein above in the context ofPDE4D7 as prostate cancer marker.

In another embodiment the present invention relates to a method fordetecting, diagnosing, monitoring or prognosticating prostate cancer orthe progression of prostate cancer comprising at least the steps of:

(a) testing in at least one sample obtained from at least one individualsuspected to suffer from prostate cancer for expression of the PDE4D7expression product or the PDE4D7 protein;

(b) testing in at least one control sample obtained from at least oneindividual not suffering from cancer for the expression of the PDE4D7expression product or the PDE4D7 protein;

(c) determining the difference in the expression of steps (a) and (b);and

(d) deciding on the presence or stage of prostate cancer or theprogression of prostate cancer based on the results obtained in step(c).

In one embodiment, steps a), b), c) and/or d) of this method ofdiagnosis may be performed outside the human or animal body, e.g. insamples obtained from a patient or individual.

In another aspect the present invention relates to a method fordiagnosing, monitoring or prognosticating hormone-resistant prostatecancer or the progression towards hormone-resistant prostate cancer,wherein said method discriminates between a hormone-sensitive and ahormone-resistant prostate cancer, comprising the steps of

(a) determining the level of PDE4D7 in a sample;

(b) determining the level of expression of a reference gene in a sample;

(c) normalizing the measured expression level of PDE4D7 to theexpression of the reference gene; and

(d) comparing the normalized expression level with a predeterminedcutoff value chosen to exclude hormone-sensitive prostate cancer,wherein a normalized expression level below the cutoff value isindicative of a hormone-resistant prostate cancer. wherein said cutoffvalue between about 1 and 7, preferably about 5.

The level of PDE4D7 may be determined on the nucleic acid, protein oractivity level as described herein above. Preferred is the determinationof the amount of PDE4D7 transcript(s) and/or protein. In addition thelevel of a reference gene in a sample may be determined. The term“reference gene” as used herein refers to any suitable gene, e.g. to anysteadily expressed and continuously detectable gene, gene product,expression product, protein or protein variant in the organism ofchoice. The term also includes gene products such as expressed proteins,peptides, polypeptides, as well as modified variants thereof. Theinvention hence also includes reference proteins derived from areference gene. Also encompassed are all kinds of transcripts derivablefrom the reference gene as well as modifications thereof or secondaryparameters linked thereto. Alternatively or additionally, otherreference parameters may also be used for reference purposes, e.g.metabolic concentrations, cell sizes etc.

The expression may be preferably be carried out in the same sample, i.e.the level of PDE4D7 and of the reference gene is determined in the samesample. If the testing is carried out in the same sample, a singledetection or a multiplex detection approach as described herein may beperformed. Preferably, for a multiplex detection the oligonucleotidesand probes having the sequence of SEQ ID NO: 7, 8 and 9 may be used. Forthe performance of the multiplex detection the concentration of primersand/or probe oligonucleotides may be modified. Furthermore, theconcentration and presence of further ingredients like buffers, ionsetc. may be modified, e.g. increased or decreased in comparison tomanufacturers' indications.

In a specific embodiment of the present invention, the expression ofmore than one reference gene or steadily expressed gene may bedetermined. E.g. the expression of 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15,20, 30 or more reference genes may be determined. The results of suchmeasurements may be either calculated separately, or may be combined inorder to obtain an average expression index. Furthermore, pattern ofreference gene expression may be determined and/or used as basis forsubsequent steps. Such pattern may be based on known expressionbehaviors of genes in certain cancer, in particular prostate cancerstages or states.

Furthermore, expression results may be compared to already known resultsfrom reference cases or databases. The comparison may additionallyinclude a normalization procedure in order to improve the statisticalrelevance of the results.

In an alternative embodiment of the present invention, instead ofdetermining the level of expression of a reference gene in a sample, theexpression of a further cancer marker or non-steadily expressed gene maybe determined. For example, the expression of a gene, which is known tobe reduced during hormone-resistant prostate cancer, or which is knownto be increased during hormone-sensitive prostate cancer, may bedetermined.

In a further embodiment, also both expression determinations may becarried out, i.e. the determination of expression of a reference geneand of a further cancer or biomarker gene.

Expression results may be normalized according to any suitable methodknown to the person skilled in the art, e.g. according to normalizationstatistical methods like the standard score, Student's T-test,studentized residual test, standardized moment text, or coeffizientvariation test. Typically, such tests or corresponding formula, whichwould be known to the person skilled in the art, would be used tostandardize expression data to enable differentiation between realvariations in gene expression levels and variations due to themeasurement processes.

Based on the expression results obtained in steps (a) and (b) and/or thenormalized results obtained in step (c) a comparison with a cutoff valuefor PDE4D7 expression may be carried out. The cutoff value below whichthe expression level of PDE4D7 is indicative of a hormone-resistantprostate cancer, thereby excluding hormone-sensitive prostate cancer ortumor forms, is between about 0.75 and 8, 0.75 and 7.5, 0.75 and 7, 0.75and 6.5, 0.75 and 6, 0.75 and 6, 0.75 and 5.5, 0.75 and 5.5, 1.0 and 8,1.25 and 8, 1.5 and 8, 1.75 and 8, 2 and 8, 2.25 and 8, 2.5 and 8, 2.75and 8, 3 and 8, 3.25 and 8, 3.5 and 8, 3.75 and 8, 4 and 8, 4.25 and 8,4.5 and 8, 4.75 and 8 or 5 and 8. More preferred is a cutoff value ofabout 5, e.g. 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1 or 5.9, 5.8,5.7, 5.6, 5.5, 5.4, 5.3, 5.2, or 5.1. In a particularly preferredembodiment, said cutoff is to be used with a housekeeping gene asreference gene. Even more preferably, said cutoff is to be used withGAPDH and/or PBGD as reference gene.

In a preferred embodiment of the present invention the cutoff value is acutoff value for PDE4D7 in blood samples, e.g. serum or plasma samples,urine samples or urine sediment samples. In a particularly preferredembodiment of the present invention the cutoff value is a cutoff valuefor the PDE4D7 protein or polypeptide or any derivative thereof asdefined herein above in a urine sample. In another particularlypreferred embodiment of the present invention the cutoff value is acutoff value for the PDE4D7 protein or polypeptide or any derivativethereof as defined herein above in cells contained in urine or exosomessecreted from cells contained in urine. In an even more preferredembodiment of the present invention the cutoff value is a cutoff valuefor the PDE4D7 protein or polypeptide or any derivative thereof asdefined herein above in a urine sediment sample and cells contained in aurine sediment sample, or exosomes secreted from cells contained in aurine sediment sample. If the measured and/or normalized PDE4D7expression is above the indicated cutoff value this may be seen as anindication that the individual is does not suffer from ahormone-resistant prostate cancer. The value may additionally indicatethat the individual suffers from a prostate cancer other thanhormone-resistant prostate cancer, in particular hormone-dependentprostate cancer or hormone-sensitive prostate cancer.

In another aspect the present invention relates to a method of dataacquisition comprising at least the steps of:

(a) testing in an individual for expression of PDE4D7; and

(b) comparing the expression as determined in step (a) to a controllevel.

The testing for expression of PDE4D7 may be carried out according tosteps as defined herein above. Preferably the testing may be carried outas measurement of nucleic acid or protein levels of PDE4D7 or bydetermining the biological activity of PDE4D7, more preferably accordingto the herein above described options for such measurements. The testingmay be carried out in an individual, i.e. in vivo, or outside theindividual, i.e. ex vivo or in vitro. The term “control level” as usedin the context of the method of data acquisition refers to theexpression of the PDE4D7 marker or other suitable markers in a cancerouscontrol or non-cancerous control, as defined herein above. The status,nature, amount and condition of the control level may be adjustedaccording to the necessities. Preferably a non-cancerous control levelmay be used. More preferably, a control level derived fromhormone-sensitive prostate cancer stages may be used. A comparison ofthe expression to a control level may be carried out according to anysuitable method of assessing, calculating, evaluating or processing ofdata and particularly aims at the detection of differences between twodata sets. A statistical evaluation of the significance of thedifference may further be carried out. Suitable statistical methods areknown to the person skilled in the art. Obtained data and informationmay be stored, accumulated or processed by suitable informatics orcomputer methods or tools known to the person skilled in the art and/orbe presented in an appropriate manner in order to allow the practitionerto use the data for one or more subsequent deduction or conclusionsteps.

In another aspect the present invention relates to an immunoassay fordetecting, diagnosing, monitoring or prognosticating prostate cancer orthe progression of prostate cancer comprising at least the steps of:

(a) testing in a sample obtained from an individual for the expressionof PDE4D7,

(b) testing in a control sample for the expression of PDE4D7,

(c) determining the difference in expression of PDE4D7 of steps (a) and(b); and

(d) deciding on the presence or stage of prostate cancer or theprogression of prostate cancer based on the results obtained in step(c).

The immunoassay is preferably based on the use of an antibodyspecifically binding to PDE4D7, e.g. one or more of the PDE4D7antibodies mentioned herein. Alternatively, the immunoassay may becarried out or combined with any other suitable agent. For example, theassay may be combined with the detection of nucleic acids, or enzymatictesting methods as described herein.

In a further aspect the present invention relates to an immunoassay fordiscriminating between a hormone-sensitive and a hormone-resistantprostate cancer, comprising the steps of

(a) determining the level of PDE4D7 in a sample;

(b) determining the level of expression of a reference gene in a sample;

(c) normalizing the measured expression level of PDE4D7 to theexpression of the reference gene; and

(d) comparing the normalized expression level with a predeterminedcutoff value chosen to exclude hormone-sensitive prostate cancer,wherein a normalized expression level below the cutoff value isindicative of a hormone-resistant prostate cancer, wherein said cutoffvalue is between about 1 and 7. Preferably, the cutoff value is about 5.

The level of PDE4D7 may preferably be determined on the protein oractivity level as described herein above. Preferred is the determinationof the amount of PDE4D7 protein with the help of PDE4D7 specificantibodies, e.g. one or more of the PDE4D7 antibodies mentioned herein.Alternatively, the immunoassay may be carried out with any othersuitable agent or be combined with the determination of other entities.For example, the assay may be combined with the detection of thepresence or amount of nucleic acids, or enzymatic testing methods asdescribed herein.

In addition the level of a reference gene as defined herein above in asample may be determined. For the detection of a reference gene theamount of the gene's expression product (i.e. protein) may bedetermined, preferably with the help of one or more suitable antibodiesknown to the person skilled in the art. Alternatively, the determinationof the reference gene may be carried out with any other suitable agentor be combined with the detection of the presence or amount of nucleicacids, or enzymatic testing methods as described herein.

Based on the expression results obtained in steps (a) and (b) and/or thenormalized results obtained in step (c) a comparison with a cutoff valuefor PDE4D7 expression may be carried out. The cutoff value below whichthe expression level of PDE4D7 is indicative of a hormone-resistantprostate cancer, thereby excluding hormone-sensitive prostate cancer ortumor forms in the immunoassay is between about 0.75 and 8, 0.75 and7.5, 0.75 and 7, 0.75 and 6.5, 0.75 and 6, 0.75 and 6, 0.75 and 5.5,0.75 and 5.5, 1.0 and 8, 1.25 and 8, 1.5 and 8, 1.75 and 8, 2 and 8,2.25 and 8, 2.5 and 8, 2.75 and 8, 3 and 8, 3.25 and 8, 3.5 and 8, 3.75and 8, 4 and 8, 4.25 and 8, 4.5 and 8, 4.75 and 8 or 5 and 8. Morepreferred is a cutoff value of about 5, e.g. 4.9, 4.8, 4.7, 4.6, 4.5,4.4, 4.3, 4.2, 4.1 or 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, or 5.1

The cutoff value may be a cutoff value for PDE4D7 in blood samples, e.g.serum or plasma samples, urine samples or urine sediment samples etc. asdescribed herein below.

If the measured and/or normalized PDE4D7 expression is above theindicated cutoff value this may be seen as an indication that theindividual is does not suffer from a hormone-resistant prostate cancer.The value may additionally indicate that the individual suffers from aprostate cancer other than hormone-resistant prostate cancer, inparticular hormone-dependent prostate cancer or hormone-sensitiveprostate cancer.

In a further aspect the present invention relates to a method ofidentifying an individual for eligibility for prostate cancer therapycomprising:

(a) testing in a sample obtained from an individual for the expressionof PDE4D7;

(b) testing in said sample for the expression of a reference gene and/ortesting in a control sample for the expression of PDE4D7;

(c) classifying the levels of expression of step (a) relative to levelsof step (b); and

(d) identifying the individual as eligible to receive a prostate cancertherapy where the individual's sample is classified as having a reducedlevel of PDE4D7 expression.

The level of PDE4D7 may be determined on the nucleic acid, protein oractivity level as described herein above. Preferred is the determinationof the amount of PDE4D7 transcript(s) and/or protein. In addition thelevel of a reference gene as described herein above in a sample may bedetermined. Testing for the expression of a reference gene may becarried out in the same sample used for the determination of PDE4D7. Ifthe testing is carried out in the same sample, a single detection or amultiplex detection approach may be performed. Preferably, for amultiplex detection the oligonucleotides and probes having the sequenceof SEQ ID NO: 7, 8 and 9 may be used. For the performance of themultiplex detection the concentration of primers and/or probeoligonucleotides may be modified. Furthermore, the concentration andpresence of further ingredients like buffers, ions etc. may be modified,e.g. increased or decreased in comparison to manufacturers' indications.Alternatively, the testing for the expression of a reference gene may becarried out in a different sample, preferably a control sample asdefined herein above. Preferably, such a control sample may be a controlsample from the same individual as the test sample, or a control samplederived from a different source or individual. The control sample mayfurther be either a sample derived from the same tissue, preferablyprostate tissue, or be derived from a different tissue type. Examples ofpreferred alternative tissue types are stromal prostate tissue, bladderepithelial tissue and urethra epithelial tissue.

Furthermore, the testing of the test sample for the expression of areference gene and the testing of control sample for the expression ofPDE4D7 may be combined.

In a further embodiment the control sample may also be tested for theexpression of the reference gene. In case more than one sample wastested for the expression of a reference gene, the obtained expressionresults may be compared and/or averaged or normalized according to anysuitable statistical method known to the person skilled in the art.

The term “classifying the levels of expression of step (a) relative tolevels of step (b)” as used herein means that the expression in a testsample for PDE4D7 and the expression in a control sample for PDE4D7 arecompared, e.g. after normalization against a suitable normalizationreferences. According to the outcome of the comparison the test sampleis indicated as providing a similar expression as the control sample, anincreased expression in comparison to the control sample, or an reducedexpression in comparison to the control sample. The term further meansthat the expression in a test sample for PDE4D7 and the expression inthe same test sample for a reference gene are compared, e.g. afternormalization against a further gene as normalization reference.According to the outcome of the comparison the test sample is indicatedas providing a similar expression as the reference gene, an increasedexpression in comparison to the reference gene, or an reduced expressionin comparison to the reference gene.

According to the classification of the expression results an individualmay be considered to be eligible for a prostate cancer therapy when thePDE4D7 expression levels are reduced. The expression level is deemed tobe “reduced” when the PDE4D7 gene expression in the test sample isdecreased by, for example, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%,40%, 50%, or more than 50% in comparison to the PDE4D7 expression in acontrol sample, or at least 0.1 fold, at least 0.2 fold, at least 1fold, at least 2 fold, at least 5 fold, or at least 10 fold or more incomparison to the PDE4D7 expression in a control sample; or when thePDE4D7 gene expression is decreased by, for example, 5%, 6%, 7%, 8%, 9%,10%, 15%, 20%, 25%, 30%, 40%, 50%, or more than 50% in comparison to theexpression of a reference gene in a control sample, or at least 0.1fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5fold, or at least 10 fold or more decreased in comparison to theexpression of a reference gene. In a specific embodiment, the expressionof a reference gene may also be normalized or adjusted to the expressionof additional genes or markers, e.g. housekeeping genes.

In a further aspect the present invention relates to an immunoassay forstratifying an individual or cohort of individuals with a prostatecancer disease comprising:

(a) testing in a sample obtained from an individual for the expressionof PDE4D7;

(b) testing in said sample for the expression of a reference gene and/ortesting in a control sample for the expression of PDE4D7;

(c) determining the difference in expression of PDE4D7 of step (a) andthe expression of PDE4D7 and/or the reference gene in step (b); and

(d) stratifying an individual or cohort of individuals to prostatecancer therapy based on the results obtained in step (c), where theindividual's sample has a reduced level of PDE4D7 expression.

The testing of the expression of PDE4D7 may preferably be carried outvia the determination of the amount of PDE4D7 protein or thedetermination of the PDE4D7 activity level as described herein above.Preferred is the determination of the amount of PDE4D7 protein with thehelp of PDE4D7 specific antibodies, e.g. one or more of the PDE4D7antibodies mentioned herein. Alternatively, the immunoassay may becarried out with any other suitable agent or be combined with thedetermination of other entities. For example, the assay may be combinedwith the detection of the presence or amount of nucleic acids, orenzymatic testing methods as described herein. In addition the level ofa reference gene as described herein above in a sample may bedetermined. Testing for the expression of a reference gene may becarried out in the same sample used for the determination of PDE4D7. Ifthe testing is carried out in the same sample, a single detection or aparallel or multiplex detection approach may be performed. Preferably,for a parallel or multiplex detection differently labeled primary orsecondary antibodies may be used.

Alternatively, the testing for the expression of a reference gene may becarried out in a different sample, preferably a control sample asdefined herein above. Preferably, such a control sample may be a controlsample from the same individual as the test sample, or a control samplederived from a different source or individual. The control sample mayfurther be either a sample derived from the same tissue, preferablyprostate tissue, or be derived from a different tissue type. Examples ofpreferred alternative tissue types are stromal prostate tissue, bladderepithelial tissue and urethra epithelial tissue. Furthermore, thetesting of the test sample for the expression of a reference gene andthe testing of control sample for the expression of PDE4D7 may becombined.

In a further embodiment the control sample may also be tested for theexpression of the reference gene. In case more than one sample wastested for the expression of a reference gene, the obtained expressionresults may be compared and/or averaged or normalized according to anysuitable statistical method known to the person skilled in the art.

The term “determining the difference in expression of PDE4D7 of step (a)and the expression of PDE4D7 and/or the reference gene in step (b)” asused herein means that the expression in a test sample for PDE4D7 andthe expression in a control sample for PDE4D7 are compared, e.g. afternormalization against a suitable normalization references. According tothe outcome of the comparison the test sample is indicated as providinga similar expression as the control sample, an increased expression incomparison to the control sample, or an reduced expression in comparisonto the control sample. The term further means that alternatively oradditionally the expression in a test sample for PDE4D7 and theexpression in the same test sample for a reference gene are compared,e.g. after normalization against a further gene as normalizationreference. According to the outcome of the comparison the test sample isindicated as providing a similar expression as the reference gene, or adifference in the expression. The difference may be either an increasedexpression in comparison to the reference gene, or a reduced expressionin comparison to the reference gene.

The term “stratifying an individual or cohort of individuals to prostatecancer therapy” as used herein means that an individual is identified aspertaining to a group of similar individuals, whose optimal therapy formis a prostate cancer therapy, preferably a therapy againsthormone-resistant prostate cancer in accordance with the outcome of theexpression test as described herein above, in particular in accordancewith encountered difference in the PDE4D7 expression level and areference gene or the PDE4D7 expression level in different samples.According to the determination of the expression difference anindividual may be identified as pertaining to a group of similarindividuals whose optimal therapy form is prostate cancer therapy whenthe PDE4D7 expression levels are reduced. The expression level is deemedto be “reduced” when the PDE4D7 gene expression in the test sample isdecreased by, for example, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%,40%, 50%, or more than 50% in comparison to the PDE4D7 expression in acontrol sample, or at least 0.1 fold, at least 0.2 fold, at least 1fold, at least 2 fold, at least 5 fold, or at least 10 fold or more incomparison to the PDE4D7 expression in a control sample; or when thePDE4D7 gene expression is decreased by, for example, 5%, 6%, 7%, 8%, 9%,10%, 15%, 20%, 25%, 30%, 40%, 50%, or more than 50% in comparison to theexpression of a reference gene in a control sample, or at least 0.1fold, at least 0.2 fold, at least 1 fold, at least 2 fold, at least 5fold, or at least 10 fold or more decreased in comparison to theexpression of a reference gene. In a specific embodiment, the expressionof a reference gene may also be normalized or adjusted to the expressionof additional genes or markers, e.g. housekeeping genes.

An individual being considered to be eligible for a prostate cancertherapy or being stratified to prostate cancer therapy as describedherein above may receive any suitable therapeutic prostate cancertreatment known to the person skilled the art. Typically, an individualconsidered to be eligible for prostate cancer therapy, or stratified toa corresponding treatment group, due to reduced PDE4D7 expression may bedeemed to be suffering from a hormone-resistant prostate cancer or beprone to develop a hormone-resistant prostate cancer in the future, e.g.within the next 1 to 24 months. A correspondingly identified orstratified individual may be treated with a pharmaceutical compositionaccording to the present invention, e.g. as defined herein below. In afurther embodiment a correspondingly identified individual may betreated with a pharmaceutical composition according to the presentinvention in combination with an additional cancer therapy. The term“additional cancer therapy” refers to any types of cancer therapy knownto the person skilled in the art. Preferred are cancer therapy formsknown for hormone-resistant prostate cancer. The term includes, forexample, all suitable forms of chemotherapy, radiation therapy, surgery,antibody therapies etc.

Alternatively, a correspondingly identified or stratified individual mayalso be treated solely with one or more cancer therapies such as achemotherapy, radiation therapy, surgery, antibody therapies etc.Preferred are cancer therapies typically used for prostate cancer, morepreferred cancer therapies used for hormone-resistant prostate cancer.

In a further embodiment of the present invention the classificationmethod for eligibility or the immunoassay for stratification asdescribed herein above may also be used for monitoring the treatment ofan individual, e.g. an individual being classified as suffering from ahormone-resistant prostate cancer. The monitoring process may be carriedout as expression determination over a prolonged period of time, e.g.during or after treatment sessions, for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10weeks, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 months, or 1, 2, 3 or more years.The determination steps may be carried out in suitable intervals, e.g.every week, 2 weeks, 3 weeks, every month, 2 months, 3 months, 6 months,12 months etc. In a further embodiment of the present invention anytreatment scheme as mentioned herein above may be adjusted, e.g.enforced or attenuated, or altered in any suitable manner incorrespondence with the results of the monitoring process.

The testing for expression of PDE4D7 may be carried out according tosteps as defined herein above. Preferably, the testing may be carriedout as measurement of protein levels of PDE4D7, more preferablyaccording to the herein above described options for such measurements.As controls or control samples controls as defined herein above may beused.

In a particularly preferred embodiment the testing steps may be based onthe use of an antibody specifically binding to PDE4D7, e.g. acommercially available anti-PDE4D7 antibody like NB300-652 or GTX14629.A cancer may be diagnosed or prognosticated or a progression of cancermay be diagnosed or prognosticated in said immunoassay or an individualmay be identified for eligibility for prostate cancer, or an individualor cohort of individuals may be stratified in an immunoassay accordingto the corresponding definitions provided herein above in the context ofthe PDE4D7 as cancer marker. Accordingly, said testing or determining ofthe expression of PDE4D7 may be accomplished, or may additionally beaccomplished, by the measurement of nucleic acid or protein levels or bythe determination of the biological activity of PDE4D7. Similarmeasurements may be carried out with respect to the reference gene.

In a particularly preferred embodiment of the present invention thereference gene is a housekeeping gene or a different phosphodiesterase.In human organisms, examples of “housekeeping genes” include inter aliaβ-actin, glycerinaldehyde 3-phosphate dehydrogenase (GAPDH),porphobilinogen deanimase (PBGD), and ribosomal protein P1. Apart fromthese genes any other suitable gene may be used as a house-keeping gene,as long as the gene shows an expression or transcription on a steady,non-modified level, in particular during different stages of cancerdevelopment, more preferably during different stages of prostate cancerdevelopment, more preferably during the transition of hormone-sensitiveprostate cancer to hormone-resistant prostate cancer states.Particularly preferred is the gene or transcript or expression productor protein of GAPDH. Further particularly preferred is the gene ortranscript or expression product or protein of PBGD. Expression data ofa house-keeping gene may be obtained from one or more samples of thesame individual or from more individuals, e.g. 2, 3, 4, 5, 6, 7, 8, 9,10, 20, 50, 100, 1000, 5.000, 10.000 or more. Expression data may alsobe obtained from databases or from data collections available to theperson skilled in the art.

The term “different phosphodiesterase” as used herein refers to otherphosphodiesterases which are not PDE4D7. Such phosphodiestersases, to besuitable as reference genes, should be steadily expressed and provide acontinuously detectable gene product, expression product, protein orprotein variant in the organism of choice. Particularly preferred arephosphodiesterases of the PDE4D family, e.g. PDE4D1, PDE4D2, PDE4D3,PDE4D4, PDE4D5, PDE4D6, PDE4D8 and PDE4D9. More preferred is the PDE4D5phosphodiesterase.

Accordingly normalization and/or comparison with GAPDH, PBGD and inparticular PDE4D5 may preferably be used for the above described cutoffbased diagnosis methods and immunoassays, the methods of identifying orthe immunoassays for discriminating or stratifying individuals.Corresponding determination steps may either be carried out in separatereactions, or, particularly preferred in multiplex reactions. For theperformance of the multiplex detection the concentration of primersand/or probe oligonucleotides may be modified. Furthermore, theconcentration and presence of further ingredients like buffers, ionsetc. may be modified, e.g. increased or decreased in comparison tomanufacturers' indications. In a further embodiment of the presentinvention the method of identifying an individual for eligibility forprostate cancer therapy based on the expression of PDE4D7 as describedherein above may further be combined with one or more similaridentification methods, based on the expression of one or more differentbiomarkers. Preferred is the determination of the level of prostatespecific antigen (PSA). Thus, if the level of PSA is encountered to 10,20, 30 or preferably above 5.0, an individual may be considered to besuffering from hormone-resistant prostate cancer, or be likely todevelop hormone-resistant prostate cancer in the near future, i.e.within the next 1, 2, 3, 4, 5, 6, 12 months

In a preferred embodiment of the present invention the diagnosing,detecting, monitoring or prognosticating as mentioned above is to becarried out on a sample obtained from an individual. The term “sampleobtained from an individual” as used herein relates to any biologicalmaterial obtained via suitable methods known to the person skilled inthe art from an individual. The sample used in the context of thepresent invention should preferably be collected in a clinicallyacceptable manner, more preferably in a way that nucleic acids (inparticular RNA) or proteins are preserved.

The biological samples may include body tissues and fluids, such asblood, sweat, sputum or saliva, semen and urine, as well as feces orstool samples. Furthermore, the biological sample may contain a cellextract derived from or a cell population including an epithelial cell,preferably a cancerous epithelial cell or an epithelial cell derivedfrom tissue suspected to be cancerous. Even more preferably thebiological sample may contain a cell population derived from a glandulartissue, e.g. the sample may be derived from the prostate of a maleindividual. Additionally, cells may be purified from obtained bodytissues and fluids if necessary, and then used as the biological sample.

Samples, in particular after initial processing, may be pooled. However,also non-pooled samples may be used.

In a specific embodiment of the present invention the content of abiological sample may also be submitted to an enrichment step. Forinstance, a sample may be contacted with ligands specific for the cellmembrane or organelles of certain cell types, e.g. prostate cells,functionalized for example with magnetic particles. The materialconcentrated by the magnetic particles may subsequently be used fordetection and analysis steps as described herein above or below.

In a specific embodiment of the invention, biopsy or resections samplesmay be obtained and/or used. Such samples may comprise cells or celllysates.

Furthermore, cells, e.g. tumor cells, may be enriched via filtrationprocesses of fluid or liquid samples, e.g. blood, urine, sweat etc. Suchfiltration processes may also be combined with enrichment steps based onligand specific interactions as described herein above.

In a particularly preferred embodiment of the present invention a samplemay be a tissue sample, a urine sample, a urine sediment sample, a bloodsample, a saliva sample, a semen sample, a sample comprising circulatingtumor cells, or a sample containing prostate secreted exosomes.

In yet another aspect the present invention relates to a pharmaceuticalcomposition comprising at least one element selected from the groupconsisting of: (a) a compound directly stimulating or modulating theactivity of PDE4D7, preferably an allosteric agonist of PDE4D7 enzymaticactivity; (b) a compound indirectly stimulating or modulating theactivity of PDE4D7; (c) the PDE4D7 protein or a biologically activeequivalent thereof; (d) a nucleic acid encoding and expressing PDE4D7;(e) a miRNA inhibitor specific for PDE4D7 miRNAs; (f) a demethylationagent; and (g) a phosphodiesterase displacement factor, preferably apeptide, a peptidomimetic, a small molecule, an antibody or an aptamer.

The term “a compound directly stimulating or modulating the activity ofPDE4D7” as used herein refers to a compound which is capable ofincreasing the activity of PDE4D7 to degrade cAMP by a directinteraction with PDE4D7. Such a compound may be any direct interactor ofPDE4D7, which has positive influence on the catalytic activity ofPDE4D7. Such a compound may preferably be an allosteric agonist of thecatalytic activity of PDE4D7, e.g. a homotropic allosteric modulator.Preferred allosteric agonists of PDE4D7 are cAMP or cAMP analogs. Otherdirectly stimulating compounds envisaged by the present invention areions, preferably biologically active mono- and bivalent cations likeCa²⁺, Mg²⁺.

The term “a compound indirectly stimulating or modulating the activityof PDE4D7” as used herein refers to a compound which is capable ofincreasing the activity of PDE4D7 to degrade cAMP by an interaction witha direct interactor of PDE4D7 (“indirect interactor”) or via an indirectworking pathway not involving an interaction with PDE4D7. Such acompound may be any direct interactor of an interactor of PDE4D7. Theeffect conveyed by the direct interactor of an interactor of PDE4D7 maybe either positive if the interactor of PDE4D7 itself has a positiveeffect on the activity of PDE4D7, or negative, if the interactor ofPDE4D7 has a negative effect on the activity of PDE4D7. Typicallypositively working indirect interactors may stimulate the agonisticeffect of direct interactors, e.g. provoke the increase of concentrationof allosterically working compounds like cAMP or analogs thereof byinhibiting cAMP degrading processes not conferred by PDE4D7, by raisingthe cAMP production etc.

Alternatively, such positively working indirect integrators may provokea modification of the binding behavior of directly binding proteins,leading to an increased PDE4D7 activity. Typically negatively workingindirect interactors may have an inhibitory effect on inhibitors ofPDE4D7. Examples of such interactors are enzymatic activities degradingPDE4D7 inhibitors, or proteins capable of binding and quenching PDE4D7inhibitors. Alternatively, such interactors may inhibit activitiesleading to a degradation of PDE4D7, e.g. proteinase inhibitors. Furtherexamples and their implementation would be known to the person skilledin the art.

Alternatively, an indirect stimulation of the PDE4D7 activity may beconveyed by compounds activating, protecting or sustaining theexpression of the endogenous PDE4D7 gene. Examples of such compounds arePDE4D7 specific transcription factors, PDE4D7 specific mRNA stabilizingactivities or PDE4D7 splice factors. Further examples and theirimplementation would be known to the person skilled in the art.

The “PDE4D7 protein” comprised in the pharmaceutical composition may bea PDE4D7 protein as defined herein above. In particular, it may be aprotein being encoded by splice variant 7 of the human phosphodiesterasePDE4D, more preferably it may have the amino acid sequence as defined inGenbank Accession No: AF536976 (version AF536976.1, GI:22901883 as of 3Mar. 2009), and even more preferably it may have the amino acid sequenceas set forth in SEQ ID NO: 2. The “PDE4D7 protein” as used in thiscontext also comprises amino acid sequences being at least 60%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to the sequence as set forth in SEQ ID NO: 2 and amino acidsequences being encoded by nucleotide sequences being at least 60%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to the sequence as set forth in SEQ ID NO: 1. Homologousvariants of PDE4D7, in particular those mentioned above, preferably havePDE4D7 functionality, i.e. are capable of degrading cAMP. In a furtherembodiment of the invention the homologous variants of PDE4D mayadditionally or alternatively have a similar or identical localizationpattern as PDE4D7 within a cell or within a tissue type.

In a further preferred embodiment the region or homology between thehomologous variants of PDE4D7 and PDE4D7 may be confined to theC-terminal part of the protein. For instance, the homologous variant maycomprise an N-terminal domain being present in PDE4D7 and a remainder ofthe protein having a degree of homology to PDE4D7 as indicated hereinabove. The N-terminal portion of the homologous variant may compriseamino acids 1 to 120, 1 to 110, 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20 or 1 to 10 derived fromPDE4D7.

The term “biologically active equivalent of PDE4D7” as used hereinrefers to a PDE4D7 protein which is capable of performing all or amajority of PDE4D7 functions. Preferably, it relates to proteins beingcapable of degrading cAMP. In a further embodiment of the invention thebiologically active equivalents of PDE4D7 may additionally oralternatively have a similar or identical localization pattern as PDE4D7within a cell or within a tissue type. Biologically active equivalentsof PDE4D7 may also comprise PDE4D7 variants as defined herein above.

PDE4D7 or biologically active equivalents of PDE4D7 according to thepresent invention may be produced recombinantly by any suitable methodknown to the person skilled in the art. The present invention, thus,also encompasses methods for the production of PDE4D7 or biologicallyactive equivalents of PDE4D7.

Accordingly, the present invention contemplates vectors containing thepolynucleotides encoding PDE4D7 or biologically active equivalents ofPDE4D7 as defined herein above, host cells, and the production of PDE4D7or biologically active equivalents of PDE4D7 by recombinant techniques.

A suitable vector may be, for example, a phage, plasmid, viral, orretroviral vector. Retroviral vectors may be replication competent orreplication defective. In the latter case, viral propagation generallywill occur only in complementing host cells. Polynucleotides encodingPDE4D7 or biologically active equivalents of PDE4D7 may be joined to avector or carrier containing a selectable marker for propagation in ahost. A corresponding polynucleotide insert may be operatively linked toan appropriate promoter, such as the phage lambda PL promoter, the E.coli lac, trp, phoA and tac promoters, the SV40 early and late promotersand promoters of retroviral LTRs, or the PSA promoter. Other suitablepromoters are known to the person skilled in the art. The expressionconstructs may further contain sites for transcription initiation,termination, and, in the transcribed region, a ribosome binding site fortranslation. The coding portion of the transcripts expressed by theconstructs will preferably include a translation initiating codon at thebeginning and a termination codon (UAA, UGA or UAG) appropriatelypositioned at the end of the polypeptide to be translated.

The polypeptides or proteins may be glycosylated or may benon-glycosylated or may otherwise by modified. In addition, polypeptidesor proteins may also include an initial modified methionine residue, insome cases as a result of host-mediated processes. Furthermore, thepolypeptide, protein or peptide may be modified by acetylation,pegylation, hesylation, formylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, specific chemicalcleavage, proteolytic cleavage, a linkage to a cellular ligand or otherprotein or hapylation, i.e. a fusion with a glycine-rich homo-amino-acidpolymer (HAP), etc. Such modifications may be carried out by suitabletechniques known to the person skilled in the art. Additionally, thepolypeptide, peptide or variant may contain one or more non-classicalamino acids.

In addition, PDE4D7 or biologically active equivalents of PDE4D7 of theinvention can be chemically synthesized using techniques known in theart, e.g. by using a peptide synthesizer.

The “nucleic acid encoding and expressing PDE4D7” comprised in thepharmaceutical composition as defined herein above refers to anysuitable carrier element, e.g. as described herein above, comprising anexpressable PDE4D7 gene. Preferably, such a carrier element may comprisethe sequence as defined in Genbank Accession No: AF536976 (versionAF536976.1, GI:22901883 as of 3 Mar. 2009), more preferably thenucleotide sequence as set forth in SEQ ID NO: 1. Such a carrier elementmay also comprises nucleotide sequences showing a high degree ofhomology to PDE4D7, e.g. nucleic acid sequences being at least 60%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical to the sequence as set forth in SEQ ID NO: 1 or nucleic acidsequences encoding amino acid sequences being at least 60%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identicalto the sequence as set forth in SEQ ID NO: 2. Alternatively, the carriermay comprise the genomic sequence of PDE4D, preferably the sequence asdefined in ENSEMBL database entry ENSG00000113448 (VersionENSG00000113448.8, Ensembl release 53—March 2009), which corresponds toSEQ ID NO: 6, or derivable from Genbank Accession No. AC008934 (VersionAC008934.5, GI:17386235 as of 24 Mar. 2009) in combination with GenbankAccession No. AC034234 (Version AC034234.4, GI:18390182 as of 24 Mar.2009) or as derivable from Wang et al., 2003, Cell Signal., 15(9):883-91. More preferably, the carrier may comprise the genomic sequenceof PDE4D as defined in SEQ ID NO: 6.

Furthermore, biologically active equivalents of PDE4D7 as defined hereinabove may be comprised in a carrier of the present invention.

The polynucleotide encoding PDE4D7 may preferably be joined to a vectorcontaining a selectable marker for propagation in a human cell. In apreferred embodiment the polynucleotide insert may be operatively linkedto a PSA promoter.

In one embodiment of the present invention nucleic acids encoding andexpressing PDE4D7 as defined herein above may be provided via livingtherapeutics. The term “living therapeutic” means that PDE4D7 orbiologically active equivalents of PDE4D7 as defined herein above areexpressed in any suitable live carrier. Accordingly, the presentinvention relates to corresponding polynucleotides which are suitablefor expression in a living cell. The present invention also relates tovectors containing such polynucleotides, appropriate host cells, and theproduction of polypeptides by recombinant techniques in said host cells.

The term “live carrier” relates to any appropriate living host cell orvirus known to the person skilled in the art. Representative examples ofappropriate hosts include, but are not limited to, bacterial cells suchas Escherichia coli or Lactobacillus, fungal cells, such as yeast cells,protozoa, insect cells, or animal cells. Preferably, the term relates toattenuated bacteria, attenuated fungal cells or attenuated protozoa.Representative examples of appropriate viruses include viruses of thegroup of adenoviruses, retrovirues or lentiviruses, preferablyattenuated viruses of the group of adenoviruses, retroviruses orlentiviruses. In a preferred embodiment, probiotic bacterial cells, inparticular probiotic Escherichia coli or Lactobacillus cells may beused. More preferably, cells of Escherichia coli Nissle 1973 and evenmore preferably cells of Lactobacillus casei or Lactobacillus zeae 393may be used.

The “miRNA inhibitor specific for PDE4D7 miRNA” comprised in thepharmaceutical composition as defined herein above refers to a nucleicacid molecule encoding a nucleic acid sequence complementary to a PDE4D7miRNA or microRNA molecule. The term “complementary” as used hereinrefers to a perfect complementary between the miRNA inhibitor nucleicacid (sense molecule) and the miRNA (antisense molecule) without anymismatch, as well as situations in which the nucleic acid contains anybase mismatches and/or additional or missing nucleotides in comparisonto the miRNA molecule. In other embodiments, the two molecules compriseone or more base mismatches or differ in their total numbers ofnucleotides (due to additions or deletions). In further embodiments, the“complementary” miRNA inhibitor nucleic acid molecule comprises at leastten contiguous nucleotides showing perfect complementarity with asequence comprised in the miRNA molecule.

Typically miRNA inhibitor nucleic acid molecules are naturally occurringDNA- or RNA molecules or synthetic nucleic acid molecules comprising intheir sequence one or more modified nucleotides which may be of the sametype or of one or more different types.

It is, for example, envisaged by the present invention that such a miRNAinhibitor nucleic acid molecule comprises at least one ribonucleotidebackbone unit and at least one deoxyribonucleotide backbone unit.Furthermore, the miRNA inhibitor nucleic acid molecule may contain oneor more modifications of the RNA backbone into 2′-O-methyl group or2′-O-methoxyethyl group (also referred to as “2′-O-methylation”), whichprevented nuclease degradation in the culture media and, importantly,also prevented endonucleolytic cleavage by the RNA-induced silencingcomplex nuclease, leading to irreversible inhibition of the miRNA.Another possible modification, which is functionally equivalent to2′-O-methylation, involves locked nucleic acids (LNAs) representingnucleic acid analogs containing one or more LNA nucleotide monomers, asdefined herein above.

Another class of silencers of miRNA expression to be used in the contextof the present invention comprises chemically engineeredoligonucleotides named “antagomirs”, which represent single-stranded RNAmolecules conjugated to cholesterol. The molecules may comprise between19 and 25 nucleotides. Preferably, the molecule comprises 20, 21, 22, 23or 24 nucleotides. More preferably, the molecule comprises 23nucleotides (further details may be derived from Krutzfeldt et al.,2005, Nature, 438: 685-689).

In another embodiment of the present invention miRNA inhibitors asdefined herein above may be provided in the form of expression vectorsto be introduced into tissue or cells. Alternatively, such vectors mayalso be introduced in living therapeutics as defined herein above.

Typically, RNAs may be produced from transgenes provided in the form oftranfection or transient expression vectors or carriers. For instance,competitive miRNA inhibitors may be provided as transcripts expressedfrom strong promoters, containing more than one, preferably multiple,tandem binding sites to a microRNA of interest. A “microRNA sponge” asdescribed in Ebert et al., 2007, Nat. Methods, 4: 721-726 is anillustrative, non-limiting example of this technique.

The “demethylation agent” comprised in the pharmaceutical composition asdefined herein above refers to an agent capable of demethylatingchromatine structures, preferably promoter regions, more preferably thePDE4D7 promoter. Examples of demethylation agents to be used in thecontext of the present invention are 5-aza-2′-deoxycytidine and5-azacytidine, which reactivate genes inappropriately silenced bystructural chromatin changes that involve DNA methylation and which canreverse these changes and, therefore, restore principal cellularpathways. This typically results in gene re-expression and reversion ofsome aspects of the transformed state. 5-azacytidine and5-aza-2′-deoxycytidine typically inactivate DNA cytosineC5-methyltransferases through the formation of stable complexes betweenthe 5-aza-2′-deoxycytidine residues in DNA and the enzyme, therebymimicking a stable transition state intermediate when bound to themethyltransferase enzyme.

A further agent, which may be comprised in a pharmaceutical compositionaccording to the present invention, either per se or in combination with5-aza-2′-deoxycytidine and/or 5-azacytidine, is trichostatin A (TSA).

The “phosphodiesterase displacement factor” comprised in thepharmaceutical composition as defined herein above refers to a compoundwhich is capable of disturbing or disrupting the interaction ofphosphodiesterases, in particular PDE4D7, with interacting partner orinteractors. Such a process may ultimately lead to an association ofPDEs, in particular PDE4D7, with different interaction partners thanbefore and, in consequence, to a redistribution of PDEs. Such newinteraction partners may sequester PDE, in particular PDE4D7, andcorrespondingly modify cellular behaviors, e.g. provoke influences onreceptor binding or other downstream activities. Examples of proteinpartners which may be involved in such a displacement reaction and/orare capable of sequestering PDE, in particular PDE4D7 are anchoringproteins like AKAPs, scaffold proteins like DISC1, beta-arrestin orRACK1, regulatory proteins like XAP2/AIP/ARA9, cAMP binding proteinslike PKA-R subunits or EPACs or receptors like the beta1-adrenoceptor,as well as enzymes like ERK.

Preferred phosphodiesterase displacement factors are peptides,peptidomimetics, small molecules, antibodies and aptamters.

A “peptide” in the context of a phosphodiesterase displacement factorrefers to a stretch of amino acids present in or representing thephosphodiesterase molecule, in particular PDE4D7, or an interacting orsequestering protein as defined herein above. The stretch of amino acidscomprised in the peptide may have a length of 5 to 100 amino acids,preferably of 10 to 50 amino acids, more preferably of 20 to 30 aminoacids. The stretches may be entirely identical to the PDE or interactorprotein or a portion thereof or may comprise sequence variations. Forexample, the peptide sequence may comprise modified amino acid residuesat up to 25% of all positions, preferably modifications which do notchange the structural properties or the binding properties of themolecule. The amino acid sequence present in the peptide mayalternatively represent spatial domains of the PDE or interactor proteinand correspondingly comprise a juxtaposition of amino acid stretcheswhich are not adjoined in the primary sequence of the molecules.

A “peptidomimetic” in the context of a phosphodiesterase displacementfactor refers is a small protein-like chain designed to mimic a peptide.Such a peptidomimetic may arise from a modification of an existingpeptide, e.g. a peptide as defined herein above, in order to alter themolecule's properties. A peptidomimetic may arise from a modificationwhich changes the molecule's stability or binding capability. Thesemodifications typically involve changes to the peptide that will notoccur naturally. For example, a peptidomimetic according to the presentinvention may have altered peptide backbones or may comprise non-naturalamino acids. Preferably, a peptidomimetic according to the presentinvention may represent a phosphodiesterase molecule, in particularPDE4D7, or an interacting or sequestering protein as defined hereinabove.

In one embodiment of the present invention a peptidomimetic may blockthe interaction between PDE, in particular PDE4D7, and its interactor.In another embodiment of the present invention a peptidomimetic mayenhance the interaction between PDE, in particular PDE4D7, and itsinteractor.

Methods and techniques for the preparation of peptidomimetics as well asassays for the testing of peptidomimetics are known to the personskilled in the art.

A “small molecules” in the context of a phosphodiesterase displacementfactor refers to a small organic compound that is preferablybiologically active, i.e. a biomolecule, but is preferably not apolymer. Such an organic compound may have any suitable form or chemicalproperty. The compound may be a natural compound, e.g. a secondarymetabolites or an artificial compound, which has been designed andgenerated de novo. In one embodiment of the present invention a smallmolecule is capable of blocking the interaction between PDE, inparticular PDE4D7, and its interactor. In another embodiment of thepresent invention a small molecule may enhance the interaction betweenPDE, in particular PDE4D7, and its interactor. Methods and techniquesfor the identification and preparation of small molecules as well asassays for the testing of small molecules are known to the personskilled in the art.

An “antibody” or an “aptamer” in the context of a phosphodiesterasedisplacement factor refers to a PDE4D7 specific antibody or antibodyvariant or fragment as defined herein above, or to a PDE4D7 specificaptamer as defined herein above, having the capability of disturbing ordisrupting the interaction between PDE, in particular PDE4D7, and one ormore of its interactors. Alternatively, the terms may also refer toantibodies or aptamers binding to any one or more of the PDE4D7interactors as described herein above, having likewise the capability ofdisturbing or disrupting the interaction between PDE, in particularPDE4D7, and one or more of its interactors. Methods for the productionor testing of antibodies or aptamers have been described herein aboveand/or are known to the person skilled in the art.

In an embodiment of the invention the pharmaceutical composition mayfurther comprise additional compounds being active against cancer cells,e.g. cytotoxic compounds or other chemotherapeutic or radiotherapeuticcompounds as known to the person skilled in the art. In a furtherembodiment the present invention also envisages screening procedures andmethods for the identification of an aptamer specific for the PDE4D7expression product or protein, a compound directly stimulating ormodulating the activity of PDE4D7, an allosteric agonist of PDE4D7enzymatic activity, a miRNA inhibitor specific for PDE4D7 miRNA, anantagomir, a PDE4D7 specific demethylation agent, a PDE4D7 specificphosphodiesterase displacement factor, a PDE4D7 specific peptidomimetic,and a PDE4D7 specific small molecule or drug as defined herein above.Such screening procedures may comprise the steps of (a) producing cellswhich express the PDE4D7 as a polypeptide either as secreted protein oron the cell membrane or as intracellular component, (b) contacting thepolypeptide produced in step (a) with a test sample potentiallycontaining an interacting molecule, e.g. an aptamer specific for thePDE4D7 protein, a compound directly stimulating or modulating theactivity of PDE4D7, a compound directly stimulating or modulating theactivity of PDE4D7, an allosteric agonist of PDE4D7 enzymatic activity,a PDE4D7 specific phosphodiesterase displacement factor, a PDE4D7specific peptidomimetic or a PDE4D7 specific small molecule or drug; and(c) indentifying an interacting molecule by observing binding and/orinhibition or modulation of the activity of PDE4D7.

Alternatively, such screening procedures may comprise the steps of (a)contacting a test sample potentially containing a directly or indirectlyinteracting molecule, e.g. an aptamer specific for the PDE4D7transcript, a miRNA inhibitor specific for PDE4D7 miRNA, an antagomir, aPDE4D7 specific demethylation agent, a PDE4D7 specific phosphodiesterasedisplacement factor, a PDE4D7 specific peptidomimetic or a PDE4D7specific small molecule or drug with one or more cells which express thePDE4D7 as a transcript, (b) detecting the expression level of saidsequence; and (c) indentifying an interacting molecule by observingbinding or a modulation or reduction of the expression level of PDE4D7.

The present invention also encompasses an aptamer specific for thePDE4D7 expression product or protein, a compound directly stimulating ormodulating the activity of PDE4D7, an allosteric agonist of PDE4D7enzymatic activity, a miRNA inhibitor specific for PDE4D7 miRNA, anantagomir, a PDE4D7 specific demethylation agent, a PDE4D7 specificphosphodiesterase displacement factor, a PDE4D7 specific peptidomimetic,and a PDE4D7 specific small molecule or drug obtainable or obtained by ascreening procedure or method as described herein above.

In a further aspect the present invention relates to a pharmaceuticalcomposition as defined herein above for the treatment or prevention ofcancer, in particular for the treatment of prostate cancer, preferablyfor the treatment of hormone-resistant prostate cancer.

Further, in yet another aspect, the present invention relates to the useof (a) a compound directly stimulating or modulating the activity ofPDE4D7, preferably an allosteric agonist of PDE4D7 enzymatic activity;(b) a compound indirectly stimulating or modulating the activity ofPDE4D7; (c) the PDE4D7 protein or a biologically active equivalentthereof; (d) a nucleic acid encoding and expressing PDE4D7; (e) a miRNAinhibitor specific for PDE4D7 miRNAs; (f) a demethylation agent; and/or(g) a phosphodiesterase displacement factor, preferably a peptide, apeptidomimetic, a small molecule, an antibody or an aptamer for thepreparation of a pharmaceutical composition for the treatment orprevention of cancer, in particular prostate cancer, preferably thetreatment of hormone-resistant prostate cancer.

In another aspect the present invention relates to a method of treatmentor prevention of cancer, in particular prostate cancer, preferably thetreatment of hormone-resistant prostate cancer, comprising theadministration of (a) a compound directly stimulating or modulating theactivity of PDE4D7, preferably an allosteric agonist of PDE4D7 enzymaticactivity; (b) a compound indirectly stimulating or modulating theactivity of PDE4D7; (c) the PDE4D7 protein or a biologically activeequivalent thereof; (d) a nucleic acid encoding and expressing PDE4D7;(e) a miRNA inhibitor specific for PDE4D7 miRNAs; (f) a demethylationagent; and/or (g) a phosphodiesterase displacement factor, preferably apeptide, a peptidomimetic, a small molecule, an antibody or an aptamerto an individual, in particular to an individual suffering from canceror being prognosticated to develop cancer.

A pharmaceutical composition according to the present invention may beadministered to a patient, subject or individual with the help ofvarious delivery systems known to the person skilled in the art, e.g.,via encapsulation in liposomes, microparticles, microcapsules,recombinant cells capable of expressing the compound, receptor-mediatedendocytosis, construction of a nucleic acid as part of a retroviral orother vector, etc. Methods of introduction may be topical, enteral orparenteral and may include intradermal, intramuscular, intraperitoneal,intravenous, subcutaneous, intranasal, inhalational, epidural, and oralroutes. The composition may be administered by any convenient route, forexample by infusion or bolus injection, by absorption through epithelialor mucocutaneous linings (e.g., oral mucosa, rectal and intestinalmucosa, etc.) or by inhalation and may be administered together withother biologically active agents. Administration can be systemic orlocal. A preferred method of local administration is by directinjection.

In another embodiment the pharmaceutical composition may be delivereddirectly to internal organs, body cavities and the like by use ofimaging devices used to guide an injecting needle directly to the siteof interest. The pharmaceutical composition may also be administered todisease sites at the time of surgical intervention. In yet anotherembodiment, the composition can be delivered in a controlled releasesystem.

Preferably the pharmaceutical composition is in a form, which issuitable for oral, local or systemic administration. In a preferredembodiment the pharmaceutical composition is administered locally,orally or systemically.

In a specific embodiment of the present invention the pharmaceuticalcomposition may be administered after an immunoassay for stratifying anindividual, or a method of identifying an individual for eligibility forprostate cancer as described herein above has been carried out, inparticular upon the classification of an individual as having a reducedlevel of PDE4D7.

In a further embodiment the pharmaceutical composition comprises atherapeutically effective amount of the ingredients of thepharmaceutical composition of the present invention as defined hereinabove and a pharmaceutically acceptable carrier. The term“pharmaceutically acceptable” means approved by a regulatory agency orother generally recognized pharmacopeia for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such a carrier is pharmaceutically acceptable, i.e. isnon-toxic to a recipient at the dosage and concentration employed.

Generally, the ingredients are supplied either separately or mixedtogether in unit dosage form, for example, as a dry lyophilised powderor water free concentrate in a hermetically sealed container such as anampoule or sachette indicating the quantity of active agent.

The pharmaceutical composition of the invention can be formulated asneutral or salt forms.

Preferably, the pharmaceutical composition may be administered directlyor in combination with any suitable adjuvant known to the person skilledin the art. The composition of the present invention can be administeredto an animal, preferably to a mammal. “Mammal” as used herein isintended to have the same meaning as commonly understood by one ofordinary skill in the art. Particularly, “mammal” encompasses humanbeings.

The term “administered” means administration of a therapeuticallyeffective dose of the aforementioned composition. By “therapeuticallyeffective amount” is meant a dose that produces the effects for which itis administered, preferably this effect is induction and enhancement ofPDE4D7. The exact dose will depend on the purpose of the treatment, andwill be ascertainable by one skilled in the art using known techniques.As is known in the art and described above, adjustments for systemicversus localized delivery, age, body weight, general health, sex, diet,time of administration, drug interaction and the severity of thecondition may be necessary, and will be ascertainable with routineexperimentation by those skilled in the art.

The concentration of the active ingredients or compounds of apharmaceutical composition according to the present invention may befurther adjusted to the intended dosage regimen, the intended usageduration, the exact amount and ratio of all ingredients of thecomposition and further factors and parameter known to the personskilled in the art.

The active agents or compounds according to the present invention may beadministered alone or in combination with other treatments. In apreferred embodiment the pharmaceutical composition of the presentinvention may be administered in combination with an anti-hormonetreatment, e.g. an anti-androgen treatment.

The pharmaceutical composition of the present invention can alsocomprise any suitable preservative known to the person skilled in theart.

Furthermore, the preparations according to the invention may alsocomprise compounds, which have an antioxidative, free-radical scavenger,antierythematous, antiinflammatory or antiallergic action, in order tosupplement or enhance their action.

In another preferred embodiment of the present invention activecomponents of the pharmaceutical composition as defined herein above maybe fused to a suitable carrier protein, e.g. to Ig Fc receptor proteinsor polymeric Ig receptors. Preferably PDE4D7 or biologically activeequivalents thereof as defined herein above may be provided as fusionproteins. The fusion partner may be provided at the N- or C-terminus.

If the pharmaceutical composition according to the present invention isto be administered in the form of a live cell or living therapeutic asdefined herein above, transformed and prepared cells may be administeredto a patient in any suitable form known to the person skilled in theart. Preferably living therapeutics may be administered in the form of acomposition comprising a microorganism, e.g. a Lactobacillus asdescribed above, in an amount between 10² to 10¹² cells, preferably 10³to 10⁸ cells.

In a further preferred embodiment of the present invention the ratiobetween two or more ingredients in the pharmaceutical composition ormedicament may be suitably adjusted according to the skilled person'sknowledge.

Suitable assays may optionally be employed to help identify optimalratios and/or dosage ranges for ingredients of pharmaceuticalcompositions of the present invention. The precise dose and the ratiobetween the ingredients of the pharmaceutical composition as definedherein above to be employed in the formulation will, inter alia, dependon the route of administration, and the exact type of disease ordisorder, and should be decided according to the judgment of thepractitioner and each patient's circumstances. Effective doses oringredient ratios may be extrapolated from dose-response curves derivedfrom in vitro or (animal) model test systems.

A typical dose can be, for example, in the range of 0.001 to 1000 μg;however, doses below or above this exemplary range are envisioned,especially considering the aforementioned factors.

In another aspect the present invention relates to a medical kit for thetreatment or prevention of cancer, in particular prostate cancer,comprising at least one element selected from the group consisting of:(a) a compound directly stimulating or modulating the activity ofPDE4D7, preferably an allosteric agonist of PDE4D7 enzymatic activity;(b) a compound indirectly stimulating or modulating the activity ofPDE4D7; (c) the PDE4D7 protein or a biologically active equivalentthereof; (d) a nucleic acid encoding and expressing PDE4D7; (e) a miRNAinhibitor specific for PDE4D7 miRNAs; (f) a demethylation agent; and (g)a phosphodiesterase displacement factor.

A medical kit that can be used in the context of the administration ofthe pharmaceutical composition as defined herein above. In particular, akit according to the present invention may be used for the treatment orprevention of cancer, in particular prostate cancer.

The ingredients of a medical kit may, according to the presentinvention, be comprised in one or more containers or separate entities.They may preferably be formulated as pharmaceutical compositions ormedicaments, more preferably they may be formulated as has beendescribed herein above in the context of the pharmaceutical compositionsof the present invention, e.g. they may comprise suitable pharmaceuticalcarriers etc. Particularly preferred are formulations for topicaladministration as mentioned herein above in the context ofpharmaceutical compositions of the invention. The medical kit accordingto the present invention may optionally also comprise a documentationwhich indicates the use or employment of the medical kit and itscomponents. Preferably, instructions comprised in the medical kit of thepresent invention may comprise recommended treatment options, dosageregimens etc. The medical kit may also comprise an instruction leafletand/or may provide additional information on the use, dosage etc.

The medical kit of the present invention may be administered to apatient according to any suitable dosage regimen known to the personskilled in the art. The medical kit or kit components may preferably begiven once a week, more preferably 2 times, 3 times, 4 times, 5 times or6 times a week and most preferably daily and or 2 times a day or moreoften, unless otherwise indicated. During progression of the treatmentthe dosages may be given in much longer time intervals and in need canbe given in much shorter time intervals, e.g., several times a day. In apreferred case a response to the treatment may be monitored using hereindescribed methods and further methods known to those skilled in the artand dosages may accordingly be optimized, e.g., in time, amount and/orcomposition. Progress can be monitored by periodic assessment. It isalso envisaged that the medical kit is employed in co-therapyapproaches, i.e. in co-administration with other medicaments or drugs,for example antibiotics, antiviral medicaments or IgG or IgAimmunoglobulins, anticancer medicaments and, preferably, anti-hormonemedicaments, more preferably anti-androgens as mentioned herein above.

In a further, specific aspect the present invention relates to a kitcomprising ingredients for the determination of the expression of PDE4D7as defined herein above together with ingredients of a medical kit forthe treatment of prostate cancer, in particular hormone-resistantprostate cancer as defined herein above.

In a further, particularly preferred embodiment of the present inventionthe cancer to be diagnosed, detected, monitored or prognosticated orwhose progression is diagnosed, detected, monitored or prognosticated orwhich is to be treated with a pharmaceutical composition as mentionedabove or by a method of treatment according to the present invention isprostate cancer.

In another particularly preferred embodiment of the present inventionthe cancer to be diagnosed, detected, monitored or prognosticated orwhose progression is diagnosed, detected, monitored or prognosticated orwhich is to be treated with a pharmaceutical composition as mentionedabove or by a method of treatment according to the present invention ishormone-resistant prostate cancer. The term “hormone-resistant prostatecancer” means that the growth and proliferation of prostate cancer orprostate cancer cell lines is resistant to male sex hormone stimulation.The term also relates to a late prostate cancer developmental stagewhich is no longer amenable to an administration of anti-hormones,preferably anti-androgens as defined herein above.

Typically, prostate cancer progression is accompanied by a shift inreliance on endocrine controls to paracrine and eventually autocrinecontrols and that this complex process is believed to be the result ofchanges which occur at molecular levels of cellular control. Due to thepossibility of metastatic spread of tumors at this stagehormone-resistant prostate cancers are a prime target for diagnosis andtreatments according to the present invention, in particular accordingto the above provided embodiments.

The following examples and figures are provided for illustrativepurposes. It is thus understood that the example and figures are not tobe construed as limiting. The skilled person in the art will clearly beable to envisage further modifications of the principles laid outherein.

EXAMPLES Example 1 Quantitative RT-PCR Assay Human Cell Lines and HumanTissue Xenografts

From the cell lines and human tissue xenografts depicted in FIG. 1 (seealso for further details Marques et al., 2006, Eur. Urol., 49(2):245-57)RNA was isolated and transcribed by standard procedures into cDNA. Theprepared cDNA's of samples “PC346P xenograft” through “346Flu2” whichare cells lines and samples “PC295” through “PC374” which are xenograftswere tested on expression levels of PDE4D7.

qRT-PCR: Materials and Methods

RNA samples were treated with DNase to ensure there was no DNAcontamination. Prior to cDNA synthesis RNA samples were treated withDnaseI (In Vitrogen) for 30 min at 37° C. 1 μg of the RNA sample wasthen treated with Superscript Vilo (In Vitrogen) to synthesize the firststrand DNA for qPCR analysis as per manufacturer's guidelines. DNAsamples were then treated with RnaseH1 30 min at 37° C.

Resulting DNA was diluted to a final concentration of 50-60 ng/μl, ofwhich 5 μl was added to each reaction well of a 96-well optical reactionplate.

Quantitative PCR reactions were performed using an ABI Prism 7300machine in a reaction volume of 15 μl according to the followingprotocol:

7.5 μl Platinum qPCR SuperMix-UDG with ROX (InVitrogen)2.2 μl nuclease free water0.1 μl 100 pmol/μl Probe0.1 μl 100 pmol/μl Forward Primer0.1 μl 100 pmol/μl Reverse Primer

Total volume in each reaction well was 15 μl including cDNA.

The PCR itself was run over 40 cycles under the following program:

Stage Repetitions Temperature (° C.) Time 1 1 50 2 seconds 2 1 95 2minutes 3 40 95 15 seconds 60 1 minuteqRT-PCR Primers and Probes (TAQMAN)

The following oligonucleotide primers and probes were used for RT-PCR onPDE4D7:

Forward Primer 5′-TGCCTCTGAGGAAACACTAC-3′ (SEQ ID NO: 3), Reverse Primer5′-GCTGAATATTGCGACATGAAAG-3′ (SEQ ID NO: 4) giving rise to a product ofthe length 101.

As probe the sequence 5′-CCAGTAATGAAGAGGAAGACCCTTTCCGC-3′ (SEQ ID NO: 5)was used.

The probe-sets was designed to target the unique N-terminal regions ofthe PDE isoform. The amplicon was designed to be within the optimalrange for Taqman assays on ABI Prism technology. All assays wereperformed in quadruplicate to maximize data integrity. A GAPDH referenceprobe was also included to which all consecutive data were referencedagainst.

qRT-PCR: Data Analysis

A −ddCt approach was carried out in order to normalize and comparedifferent RT-PCR experiments. Ct values were obtained by manualthreshold observation where each probe-set was amplifying exponentiallyat a comparable efficiency. In particular, the following steps werecarried out:

1.) The difference in cycle number (Ct) between reference and gene ofinterest (GAPDH subtracted from Gene of interest) was calculated to givethe experimental sample (ES) dCt.2.) One sample was selected as standard to be compared against (LNCaP)(C) and its dCt was calculated.3.) The change in cycle number difference could be derived by dCt(ES)—dCt(C)=ddCt4.) The final comparable expression values could be derived by 2-ddCt inorder to take into account the doubling of DNA after each cycle, henceshowing the amount of mRNA in comparison to LNCaP.

This operation gave a value in comparison to LNCaP (which will have thevalue of 1), i.e. any value >1 was considered to be an increase inexpression, a value of <1 was considered to be a decrease in expression.

It was accordingly assumed that the extension efficiencies of all thePCR reactions are within a certain range, resulting in a value of 1.

Percentage Approach to Normalize and Compare Different RT-PCRExperiments

For each probe-set a Ct (cycle number) value was obtained. This wasgenerated by finding a baseline which intersected the amplificationcurves during their exponential phase. The baselines were generateddynamically according to the curves obtained in each experiment. The Ct(intersect or cycle) values of the GOIs were then subtracted from the Ctvalue of the GAPDH standard.

According to the formula Ct(GAPDH)−Ct(GOI)=dCt, given that GOI Ct valuesare always larger than the reference gene the dCt value resulted innegative numbers, i.e. a −dCt value.

Based on the doubling effect of each cycle and the absolute values weredetermined according to the Comparative Expression Value=2−dCt. Due tothe very small values gained from this calculation the value multipliedby 1000 for handling purposes.

Expression levels for PDE4D7 obtained by this approach are depicted inFIGS. 2 to 7. In particular, FIGS. 2 to 4 cover the relative expressionof PDE4D7 cDNA in different human prostate cancer cell lines andxenografts (see FIG. 1) normalized according to the −ddCt approach,whereas FIGS. 5 to 7 cover the relative expression of PDE4D7 cDNA indifferent human prostate cancer cell lines and xenografts (see FIG. 1)normalized relative to total PDE4D expression in the individual celllines or xenograft tissue material.

As can be derived from FIG. 2-7 the expression (or transcription) levelof PDE4D7 is significantly different between most androgen-dependent orandrogen-sensitive vs. androgen-independent cell lines (FIGS. 3 and 6)or human xenografts (FIGS. 4 and 7). FIGS. 2 and 5 show the combinedresults of cell lines and xenografts.

According to the results presented in FIGS. 2 to 7 the transcription ofPDE4D7 in human prostate cancer cell lines and tissues is dependent onthe status of the Androgen Receptor activity in a given cell type. Incase of presence of AR and sensitivity of a cell to androgen/hormonestimulation significant PDE4D7 transcription can be observed whereas onthe absence of active AR the PDE4D7 transcription is very minimal, i.e.virtually absent.

Example 2 Quantitative RT-PCR Assay with Human Tissue Samples

The relative gene expression of human PDE4D7 was evaluated in prostatecancer tissues derived from patients with hormone-sensitive/responsivevs. hormone-refractory/castration-resistant patients.

Materials and Methods

Details on the samples used in the qPCR measurements of PDE geneexpression experiment are given in Table 1, below:

TABLE 1 Patient information used in the experiment Hormone Type ofGleason Age at Refractory Tissue Tissue Score Treatment Status ProstateTURP 4 + 4 66 no Lymphnode 4 + 4 63 no Prostate TURP 3 + 3 67 noProstate TURP 4 + 4 82 no Prostate TURP 5 + 4 76 yes Prostate TURP 3 + 460 yes Lymphnode 4 + 4 66 no Prostate TURP 5 + 3 90 yes Prostate TURP4 + 3 66 yes Prostate TURP 3 + 4 60 yes Prostate TURP 4 + 4 64 noProstate TURP 6 71 yes Lymphnode 4 + 4 62 no Prostate TURP 7 75 yesProstate TURP 3 + 5 70 yes Lymphnode 4 + 4 52 no

All samples were derived from male patients (ages 52-82). The column“Tissue” defines the tissue that has been taken during surgery, eitherprostate tissue, or lymph nodes for staging. The column “Type of Tissue”describes the approach of tissue resection. If not otherwise indicatedthe tissue was resected during prostate surgery (prostatectomy). “TURP”is defined as Trans Urethral Resection of the Prostate. The cDNA panelincludes 4 samples derived from patients with hormone-sensitive prostatecancer, and 8 samples derived from patients with hormone-refractoryprostate cancer (indicated by “yes” in the column “Hormone RefractoryStatus”) and 4 samples from lymph node metastases

Primer and probe sequences used for human PDE4D7:

sense primer sequence: (SEQ ID NO: 7) CGGAATGGAACCCTA TCTTGTCantisense primer sequence: (SEQ ID NO: 8) TTGGTCGTTGAATGTTCTCTGATprobe sequence: (SEQ ID NO: 9)CCTCTCGCCTTCAGACAGTTGGAACAAGGAG AGG (FAM-labeled).

The PDE4D7 specific primers (SEQ ID NO: 7 and 8) were premixed with theFAM probes (SEQ ID NO: 9) to perform quantitative, real-time PCR (qPCR),and used in a 1:20 dilution according to manufacturer's description(PrimerDesign, UK). The human cDNA samples are arranged in standard,qPCR-ready, 96-well microtiter (MT) plates.

16 tissue samples, derived from 16 different patients were arranged per96-well MT plate, with each of the 16 wells used per plate containing ca2-3 ng of RNA reverse transcribed cDNA.

To each of the used well of the MT plate 15 μL Applied Biosystems'GeneAmp mastermix (2×), 13.5 μL RNAse/DNAse free water and 2 μLPrimerDesign PerfectProbe primermix (PrimerDesign, UK) were added.

All samples were analyzed with the following PCR protocol: 2 min at 50°C., 10 min at 95° C., 15 sec at 95° C., 30 sec at 50° C. while recordingfluorescence, 15 sec at 72° C. and the last three steps repeated 50times.

For all calculations relative gene expression values, the followingprocedure was used: C_(T) values of 40 or higher or below 16 wereexcluded for poor quality reasons. (The genes examined here had anaverage C_(T)-value of ˜33).

To normalize the C_(T) values, the following approach was used: theC_(T) values were converted to relative gene copy number based oncalibration curves. The calibration curves were independently measuredon different dilutions of cDNA. Subsequently the PDE4D7 expression bydividing the PDE4D7 copy number by the average copy number of thehousehold genes (Glycerinaldehyde-3-phosphate-Dehydrogenase (GAPDH), andPorphobilinogen Deaminase (PBGD)).

Relative Expression of Human PDE4D7 in Human Prostate Tissues(Hormone-Responsive Vs. Hormone-Resistant) Including Lymph Node ResectedTissue Samples

The gene expression level of the human PDE4D7 isoform was determined onhuman prostate tissues as described above. The relative expressionlevels were determined in two defined prostate tissues:“hormone-responsive” and “hormone-refractory”. For an initialinvestigation of the human PDE4D7 expression status lymph node resectedtissue samples together with primary prostate cancer samples wereincluded.

Lymph node resected tissue samples are characterized by invasive growthas they have been disseminating from the primary tumor into the sentinellymph nodes. In that sense these samples are from a molecularperspective different from the primary tumor cells, which arecharacterized by uncontrolled proliferation but are still growing withinthe primary organ confines. Invasive growth implicates more aggressivegrowth behavior as also found in the hormone-refractory prostate cancertissue.

A Student's T-test was performed to see whether human PDE4D7 geneexpression is on average slightly decreased in hormone-resistant tumortissues compared to hormone-responsive human prostate tissue.

As can be derived from FIGS. 8 and 9, the expression in thehormone-resistant prostate tumors is generally decreased but cannoteasily be distinguished from the expression in the hormone-sensitiveprostate tumor groups. The average PDE4D7 expression difference betweenthe tested tissue types was correspondingly found to be not significant(p-value=0.22).

This result could be possibly explained by the more aggressive behaviorof part of the samples included in the analysis of group 1. It has beenspeculated whether aggressive disease harbors already tumor cells thathave lost their responsiveness to androgen, such that thecharacteristics of hormone-refractory or hormone-resistant behaviorwould be inherently contained within an aggressive tumor. These cellswould consequently be selected under androgen deprivation conditions.This could explain why such tissue types already tend to have reducedexpression levels of human PDE4D7 leading to less significant p-valuesin a standard T-test. Thus, the difficulty in correctly determining thestatus of lymph node resected tissue samples, i.e. the fact that itcould not be excluded that these tumors already contained to some extenthormone-resistant tumor cell populations could possibly have lead to theobtained results.

Relative Expression of Human PDE4D7 in Human Prostate Tissues(Hormone-Responsive Vs. Hormone-Resistant) Excluding Lymph Node ResectedTissue Samples

In order to circumvent the encountered problems, the relative expressionlevels were determined in two defined prostate tissues:“hormone-responsive” and “hormone-refractory”, whereby for theinvestigation of the human PDE4D7 expression status only primaryprostate cancer samples were included, i.e. all samples derived fromlymph node resections were dismissed.

A Student's T-test was performed to see whether human PDE4D7 geneexpression is significantly decreased on average in hormone-resistanttumor tissues compared to hormone-responsive human prostate tissue.

As can be derived from FIGS. 10 and 11, the expression in thehormone-resistant prostate tumors is decreased and can easily bedistinguished from the expression in the hormone-sensitive prostatetumor groups. The average PDE4D7 expression difference between the twotissue types (hormone-resistant tumor tissues vs. hormone-sensitivetumor tissues) is significant (p=0.032).

FIGS. 10 and 11 show that the PDE4D7 expression in the hormone-resistantprostate tumors is generally decreased compared to hormone-responsiveprostate tumors. It is hence concluded that a decreased level ofcAMP-PDE activity is advantageous for enhanced cell proliferation. Ithas been long speculated that next to Androgen Receptor gene mutationsor gene amplification, the activation of other cellular signalingpathways can support the transition of hormone-responsive tohormone-independent cell growth. The cAMP-PKA pathway is one of thepathways that have been implicated in that transition of hormone relatedgrowth. The change in PDE4D7 expression from hormone-sensitive tohormone-refractory human prostate tissue supports this view.

Example 3 The Effect of PDE4D7 Expression on PC3 Proliferation

To test the effect of heterologous expression of human PDE4D7 on theproliferation of androgen-independent cell lines several gene constructswere transfected into PC3 cells and tested cell growth compared tocontrol treated cell lines. As controls untreated PC3 cells, PC3 celltransfected with a dominant negative form of PDE4D7 wherein thecatalytic activity of PDE4D7 has been knocked out by genetic mutation ofthe coding sequence of PDE4D7, as well as PC3 cells transfected with adifferent isoform of the PDE4D family, namely PDE4D1, were used.

PC3 Cells were grown at 5% CO2, 37 C, 5% Foetal Bovine Serum in RPMI1640 supplemented with L-Glutamine (2 mM final concentration) andPenStrep (1%). The PC3 cells were seeded at a density of 5000 cells perwell of a 96 well plate (MTP). The Transfection complexes for the geneof interest (PDE4D7, PDE4D7 dominant Negative and PDE4D1) were formedusing Fugene 6 Transfection reagent as per the manufacturer'sinstructions. For each gene of interest ˜25 independent transfectionswere performed to increase the statistical power of the test (each barrepresents ˜25 independent measurements).

After initial plating time of 4 hours the transfection complexes wereadded to the growth media and the cells were allowed to proliferate for60 hours before the addition of the Promega MTT compound. The MTT assayprocedure to investigate cell proliferation was followed as permanufacturer's instructions.

Absorbance readings were obtained using the Mithras LB940 plate readersystem. 1 second readings were taken from each well.

As demonstrated in the above experiment human PDE4D7 heterologousexpression in the androgen-independent prostate cancer cell line PC3showed a negative effect on proliferation in vivo. Cell proliferationunder the chosen conditions was overall inhibited ˜15% by expression ofhuman PDE4D7 compared to untreated cells, cells transfected with aninactive form of PDE4D7 as well as compared to PDE4D1 expression. Theeffect is statistically significant (p<0.001 compared to untreated PC3).

This experiment demonstrates that the activation of human PDE4D7 has aninhibitory effect on cell proliferation of androgen-independent prostatecancer cells. The observed effect on cell proliferation is statisticallyhighly significant.

Due to the transient manner of the transfection used for the experiment(i.e., depending on the transfection efficiency more or less PC3 lesswere transfected and subsequently overexpress the PDE enzyme) it isexpected that an even higher level of inhibition of cell proliferationcan be reached once a higher transient transfection efficiency isachieved in PC3 cells.

The present application comprises the following additional embodiments:

Item 1: Phosphodiesterase 4D7 (PDE4D7) for use as a marker for cancer.Item 2: A composition for diagnosing, detecting, monitoring orprognosticating cancer or the progression of cancer, comprising anucleic acid affinity ligand and/or a peptide affinity ligand for thePDE4D7 expression product or protein.Item 3: The composition of item 2, wherein said nucleic acid affinityligand or peptide affinity ligand is modified to function as a contrastagent.Item 4: The composition of item 2, wherein said affinity ligand is a setof oligonucleotides specific for the PDE4D7 expression product, a probespecific for the PDE4D7 expression product, an aptamer specific for thePDE4D7 expression product or for the PDE4D7 protein, an antibodyspecific for the PDE4D7 protein and/or an antibody variant specific forthe PDE4D7 protein.Item 5: Use of PDE4D7 as a marker for diagnosing, detecting, monitoringor prognosticating cancer or the progression of cancer.Item 6: A method for detecting, diagnosing, monitoring orprognosticating cancer or the progression of cancer comprising at leastthe step of determining the level of PDE4D7 in a sample.Item 7: The method of item 6, wherein the determining step isaccomplished by the measurement of nucleic acid or protein levels or bythe determination of the biological activity of PDE4D7.Item 8: The method of item 7, wherein said method comprises theadditional step of comparing the measured nucleic acid or protein levelsor the measured biological activity to a control level.Item 9: A method of data acquisition comprising at least the steps of:

(a) testing in an individual for expression of PDE4D7; and

(b) comparing the expression as determined in step (a) to a controllevel.

Item 10: The use of item 2 or the method of any one of items 6 to 9,wherein the diagnosing, detecting, monitoring, prognosticating or dataacquisition is to be carried out on a sample obtained from anindividual.Item 11: An immunoassay for detecting, diagnosing, monitoring orprognosticating cancer or the progression of cancer comprising at leastthe steps

(a) testing in a sample obtained from an individual for the expressionof PDE4D7,

(b) testing in a control sample for the expression of PDE4D7,

(c) determining the difference in expression of PDE4D7 of steps (a) and(b); and

(d) deciding on the presence or stage of cancer or the progression ofcancer based on the results obtained in step (c),

wherein said testing steps are based on the use of an antibodyspecifically binding to PDE4D7.

Item 12: The use or method of item 10 or the immunoassay of item 11,wherein said sample is a tissue sample, a urine sample, a urine sedimentsample, a blood sample, a saliva sample, a semen sample, or a samplecomprising circulating tumor cells.Item 13: A pharmaceutical composition comprising at least one elementselected from the group of:

(a) a compound directly stimulating or modulating the activity ofPDE4D7, preferably an allosteric agonist of PDE4D7 enzymatic activity;

(b) a compound indirectly stimulating or modulating the activity ofPDE4D7;

(c) the PDE4D7 protein or a biologically active equivalent thereof;

(d) a nucleic acid encoding and expressing PDE4D7;

(e) a miRNA inhibitor specific for PDE4D7 miRNAs;

(f) a demethylation agent; and

(g) a phosphodiesterase displacement factor, preferably a peptide, apeptidomimetic, a small molecule, an antibody or an aptamer.

Item 14: A pharmaceutical composition for the treatment or prevention ofcancer comprising at least one element selected from the group of:

(a) a compound directly stimulating or modulating the activity ofPDE4D7, preferably an allosteric agonist of PDE4D7 enzymatic activity;

(b) a compound indirectly stimulating or modulating the activity ofPDE4D7;

(c) the PDE4D7 protein or a biologically active equivalent thereof;

(d) a nucleic acid encoding and expressing PDE4D7;

(e) a miRNA inhibitor specific for PDE4D7 miRNAs;

(f) a demethylation agent; and

(g) a phosphodiesterase displacement factor preferably a peptide, apeptidomimetic, a small molecule, an antibody or an aptamer.

Item 15: Use of

(a) a compound directly stimulating or modulating the activity ofPDE4D7, preferably an allosteric agonist of PDE4D7 enzymatic activity;

(b) a compound indirectly stimulating or modulating the activity ofPDE4D7;

(c) the PDE4D7 protein or a biologically active equivalent thereof;

(d) a nucleic acid encoding and expressing PDE4D7;

(e) a miRNA inhibitor specific for PDE4D7 miRNAs;

(f) a demethylation agent; and/or

(g) a phosphodiesterase displacement factor, preferably a peptide, apeptidomimetic, a small molecule, an antibody or an aptamer,

for the preparation of a pharmaceutical composition for the treatment orprevention of cancer.

Item 16: The phosphodiesterase of item 1, the composition of any one ofitems 2 to 4, the use of item 5, 10 or 12, the method of any one of item6 to 10 or 12, the immunoassay of item 11 or 12, the pharmaceuticalcomposition of item 13 or 14, or the use of item 15, wherein said canceris prostate cancer.Item 17: The phosphodiesterase, use, composition, method, immunoassay,or pharmaceutical composition of item 16, wherein said prostate canceris hormone-resistant prostate cancer.

1. Phosphodiesterase 4D7 (PDE4D7) for use as a marker for prostatecancer.
 2. A composition for diagnosing, detecting, monitoring orprognosticating cancer or the progression of prostate cancer, comprisinga nucleic acid affinity ligand and/or a peptide affinity ligand for thePDE4D7 expression product or protein.
 3. The composition of claim 2,wherein said nucleic acid affinity ligand or peptide affinity ligand ismodified to function as a contrast agent.
 4. The composition of claim 2,wherein said affinity ligand is a set of oligonucleotides specific forthe PDE4D7 expression product, a probe specific for the PDE4D7expression product, an aptamer specific for the PDE4D7 expressionproduct or for the PDE4D7 protein, an antibody specific for the PDE4D7protein and/or an antibody variant specific for the PDE4D7 protein. 5.Use of PDE4D7 as a marker for diagnosing, detecting, monitoring orprognosticating prostate cancer or the progression of prostate cancer.6. A method for detecting, diagnosing, monitoring or prognosticatingprostate cancer or the progression of prostate cancer comprising atleast the step of determining the level of PDE4D7 in a sample.
 7. Amethod for diagnosing, monitoring or prognosticating hormone-resistantprostate cancer or the progression towards hormone-resistant prostatecancer, wherein said method discriminates between a hormone-sensitiveand a hormone-resistant prostate cancer, comprising the steps of (a)determining the level of PDE4D7 in a sample; (b) determining the levelof expression of a reference gene in a sample; (c) normalizing themeasured expression level of PDE4D7 to the expression of the referencegene; and (d) comparing the normalized expression level with apredetermined cutoff value chosen to exclude hormone-sensitive prostatecancer, wherein a normalized expression level below the cutoff value isindicative of a hormone-resistant prostate cancer, wherein said cutoffvalue is between about 1 and 7, preferably about
 5. 8. A method of dataacquisition comprising at least the steps of: (a) testing in anindividual for expression of PDE4D7; and (b) comparing the expression asdetermined in step (a) to a control level.
 9. An immunoassay fordetecting, diagnosing, monitoring or prognosticating prostate cancer orthe progression of prostate cancer comprising at least the steps (a)testing in a sample for the expression of PDE4D7, (b) testing in acontrol sample for the expression of PDE4D7, (c) determining thedifference in expression of PDE4D7 of steps (a) and (b); and (d)deciding on the presence or stage of prostate cancer or the progressionof prostate cancer based on the results obtained in step (c), whereinsaid testing steps are based on the use of an antibody specificallybinding to PDE4D7.
 10. An immunoassay for discriminating between ahormone-sensitive and a hormone-resistant prostate cancer, comprisingthe steps of (a) determining the level of PDE4D7 in a sample; (b)determining the level of expression of a reference gene in a sample; (c)normalizing the measured expression level of PDE4D7 to the expression ofthe reference gene; and (d) comparing the normalized expression levelwith a predetermined cutoff value chosen to exclude hormone-sensitiveprostate cancer, wherein a normalized expression level below the cutoffvalue is indicative of a hormone-resistant prostate cancer, wherein saidcutoff value is between about 1 and 7, preferably about
 5. 11. A methodof identifying an individual for eligibility for prostate cancer therapycomprising: (a) testing in a sample obtained from an individual for theexpression of PDE4D7; (b) testing in said sample for the expression of areference gene and/or testing in a control sample for the expression ofPDE4D7; (c) classifying the levels of expression of step (a) relative tolevels of step (b); and (d) identifying the individual as eligible toreceive a prostate cancer therapy where the individual's sample isclassified as having a reduced level of PDE4D7 expression.
 12. Animmunoassay for stratifying an individual or cohort of individuals witha prostate cancer disease comprising: (a) testing in a sample obtainedfrom an individual for the expression of PDE4D7; (b) testing in saidsample for the expression of a reference gene and/or testing in acontrol sample for the expression of PDE4D7; (c) determining thedifference in expression of PDE4D7 of step (a) and the expression ofPDE4D7 and/or the reference gene in step (b); and (d) stratifying anindividual or cohort of individuals to prostate cancer therapy based onthe results obtained in step (c), where the individual's sample has areduced level of PDE4D7 expression.
 13. The method of claim 6, whereinsaid testing or determining of the expression is accomplished, or isadditionally accomplished, by the measurement of nucleic acid or proteinlevels or by the determination of the biological activity of PDE4D7, orof the reference gene.
 14. The method or immunoassay of claim 13,wherein said method or immunoassay comprises the additional step ofcomparing the measured nucleic acid or protein levels or the measuredbiological activity to a control level.
 15. The method of claim 7,wherein said reference gene is a housekeeping gene, preferably GAPDH orPBGD, or a different phosphodiesterase, preferably PDE4D5.
 16. Themethod of claim 7, wherein said method or immunoassay comprises theadditional step of determining the level of prostate specific antigen(PSA).
 17. The method of claim 7, wherein said sample is a tissuesample, a urine sample, a urine sediment sample, a blood sample, asaliva sample, a semen sample, a sample comprising circulating tumorcells or a sample containing prostate secreted exosomes.
 18. Apharmaceutical composition comprising at least one element selected fromthe group of: (a) a compound directly stimulating or modulating theactivity of PDE4D7, preferably an allosteric agonist of PDE4D7 enzymaticactivity; (b) a compound indirectly stimulating or modulating theactivity of PDE4D7; (c) the PDE4D7 protein or a biologically activeequivalent thereof; (d) a nucleic acid encoding and expressing PDE4D7;(e) a miRNA inhibitor specific for PDE4D7 miRNAs; (f) a demethylationagent; and (g) a phosphodiesterase displacement factor, preferably apeptide, a peptidomimetic, a small molecule, an antibody or an aptamer.19. A pharmaceutical composition for the treatment or prevention ofprostate cancer comprising at least one element selected from the groupof: (a) a compound directly stimulating or modulating the activity ofPDE4D7, preferably an allosteric agonist of PDE4D7 enzymatic activity;(b) a compound indirectly stimulating or modulating the activity ofPDE4D7; (c) the PDE4D7 protein or a biologically active equivalentthereof; (d) a nucleic acid encoding and expressing PDE4D7; (e) a miRNAinhibitor specific for PDE4D7 miRNAs; (f) a demethylation agent; and (g)a phosphodiesterase displacement factor preferably a peptide, apeptidomimetic, a small molecule, an antibody or an aptamer. 20.(canceled)
 21. (canceled)